Open access

Bone and Cartilage from Stem Cells: Growth Optimalization and Stabilization of Cell Phenotypes

Written By

Jan O Gordeladze, Janne E Reseland, Tommy A Karlsen, Rune B Jakobsen, Lars Engebretsen, Ståle P Lyngstadaas, Isabelle Duroux-Richard, Christian Jorgensen and Jan E Brinchmann

Submitted: November 4th, 2010 Published: August 29th, 2011

DOI: 10.5772/19826

Chapter metrics overview

3,622 Chapter Downloads

View Full Metrics

1. Introduction

Engineered cells replacing tissues should mimic the three-dimensional (3D) structure and reflect the different cell phenotypes exhibited by the lost or damaged tissue (Raimondi 2006; Keung, Healy et al. 2010). The engineered cells should also demonstrate a certain plasticity, i.e. an ability to adapt to the environment, in which they are deposited, reflecting the minute differences in features necessary to rebuild a functional tissue, which is able to renew itself over time (Grad and Salzmann 2009; Ohishi, Chiusaroli et al. 2009; Tare, Kanczler et al. 2010).

Osteoblastic cells in bone need to be able to involve themselves in a remodelling cycle with osteoclasts (Hanada, Hanada et al. 2010; Trouvin and Goeb 2010), which may be recruited from surrounding bone structures, and/or may be furnished as preosteoclasts within the population of osteoblasts. Furthermore, the osteoblasts should be able to undergo a distinct alteration in terms of life-span defined characteristics (Lian and Stein 2003; Lian, Stein et al. 2006; Gordeladze, Reseland et al. 2009 ), including the transition to osteocytes. Finally, the newly formed engineered tissue does not survive unless it develops a vascular network (Matsumoto, Kuroda et al. 2008; Grellier, Bordenave et al. 2009 ) furnishing the bone tissue with oxygen, growth factors and nutrients.

Chondrocytes in engineered cartilage should be able to produce an extracellular matrix reflecting the composition, water-binding capacity and mechanical characteristics of true hyaline cartilage (Knecht, Vanwanseele et al. 2006; Heinegard 2009; Bertrand, Cromme et al. 2010; Goldring and Goldring 2010). This type of cartilage exhibits certain features, such as hypoxic conditions and chondrocytes demonstrating gradients of gene transcript levels (cell phenotype plasticity) between the juxta-luminal and bone-lining surfaces of a joint (Grimshaw and Mason 2001; Lu, Subramony et al. 2010; Oh, Kim et al. 2010).

The features listed above have been extensively described and confirmed in the literature, however, a joint approach to produce well adapting engineered osteoblasts and chondrocytes has hitherto not been the subject of review articles or book chapters. The present outline encompasses a combined literature review of phenomena to take into consideration when engineering such cells from stem cells (SCs): sources of SCs to use (Logeart-Avramoglou, Anagnostou et al. 2005; van Osch, Brittberg et al. 2009), genes or microRNAs to manipulate (Goldring, Tsuchimochi et al. 2006; Betz 2008; Grundberg, Brandstrom et al. 2008; Duggal, Fronsdal et al. 2009; Gordeladze, Djouad et al. 2009 ; Lin 2009; Granchi, Ochoa et al. 2010; Sun 2010 ; Herlofsen, Kuchler et al. 2011), selection of gene and microRNA delivery systems (Saraf and Mikos 2006; Phillips, Gersbach et al. 2007), choice of humoral growth factors to facilitate SC differentiation (Shahdadfar, Fronsdal et al. 2005; Boeuf and Richter 2010; van der Kraan, Davidson et al. 2010), selection of appropriate scaffolds to support “asymmetric” SC differentiation ( Vinatier, Bouffi et al. 2009 ; Oh, Kim et al. 2010; Seidi, Ramalingam et al. 2011), combination of stem cell niches and/or co-cultures to ensure organ mimicry reflecting proper cell-cell interactions (Grad and Salzmann 2009; Grellier, Bordenave et al. 2009 ; Boeuf and Richter 2010; Tare, Kanczler et al. 2010), mechano-stimulation of cells (Kelly and Jacobs 2010; Nowlan, Sharpe et al. 2010 ), and three-dimensional (3D) organ printing (Williams 2009; Visconti, Kasyanov et al. 2010).

Furthermore, this review also discusses how to stabilize osteoblasts and chondrocytes obtained by differentiation of SCs, i.e. how we can make the subject cells resilient to the detrimental effects of inflammatory cytokines and T-cells ( Gordeladze, Reseland et al. 2009 ; Gruber 2010; Hanada, Hanada et al. 2010; Pacifici 2010), and exosomes shredded from immune cells (Valadi, Ekstrom et al. 2007; Camussi, Deregibus et al. 2010; Zomer, Vendrig et al. 2010).


2. Sources of stem cells for osteoblast and chondrocyte differentiation

Osteoblast differentiation

Bone marrow, which is the natural repository of osteoblasts, is widely used as source for bone engineering. Under appropriate conditions, bone-derived stem cells (bone mesenchymal stem cells = bone MSCs) can differentiate into osteoblasts, chondrocytes, adipocytes, and stromal cells (Javazon, Beggs et al. 2004; Otto and Rao 2004; Logeart-Avramoglou, Anagnostou et al. 2005). The differentiating potency of bone MSCs was enhanced when embedded in diffusion chambers or organ capsules, however, there is now a plethora of scaffolds securing the development of “proper” osteoblasts to produce bone for tissue replacement purposes. The advantage of using bone MSCs is related to the large number of obtainable osteoblasts, their high number of passages before the differentiating potential is lost, and their ability to be stored frozen for a long period. And the default pathway of bone MSCs is the osteogenic pathway (Logeart-Avramoglou, Anagnostou et al. 2005).

During the past 10-12 years, many other stem cell sources with osteogenic potential have been isolated, including blood, adipose tissue, lung, synovium, skeletal muscle and tooth pulp (for review, (Barry and Murphy 2004 ; Logeart-Avramoglou, Anagnostou et al. 2005; Gordeladze, Reseland et al. 2009 ; Bodle, Hanson et al. 2011; Levi and Longaker 2011; Witkowska-Zimny and Walenko 2011)). However, it seems that adipose stem cells (ASCs), provided that they are similar to MSCs in terms of surface receptor molecule profile (STRO-1, CD34, CD45, CD117 negative; CD44, CD49 CD29, CD90, CD105, CD106 positive) (Logeart-Avramoglou, Anagnostou et al. 2005; Niemeyer, Krause et al. 2006), may serve as a good source for bone engineering (Bodle, Hanson et al. 2011; Levi and Longaker 2011; Monaco, Bionaz et al. 2011). Irrespective of whether the source encompasses MSCs or ASCs, it seems that “proper” osteoblasts may be obtained if incubation conditions (i.e. the choice of growth factor source) and appropriate scaffolds are employed (Logeart-Avramoglou, Anagnostou et al. 2005; Kanczler and Oreffo 2008; Kwan, Slater et al. 2008; Gordeladze, Reseland et al. 2009 ; Tiainen, Lyngstadaas et al. 2010; Rahaman, Day et al. 2011; Sabetrasekh, Tiainen et al. 2011 ).

Chondrocyte differentiation

As for bone engineering, cartilage engineering relies firmly on the use of MSCs and ASCs (van Osch, Brittberg et al. 2009; Boeuf and Richter 2010; O'Sullivan, D'Arcy et al. 2011; Witkowska-Zimny and Walenko 2011). Some other sources, i.e. ectodermal cells like skin and hair follicles, as well as perinatal tissue and umbilical cord blood (Kuhn and Tuan 2010). One article refers to the use of synovial membrane stem cells (SMSCs) and compares their potency for chondrocyte differentiation with MSCs and ASCs (Segawa, Muneta et al. 2009). The criteria for selection of cell source may vary, but the authors focus on the necessity to analyse chondrocytes differentiated from these stem cells and choose the better source depending on how close they resemble the gene expression profile of mature chondrocytes isolated from hyaline cartilage (Segawa, Muneta et al. 2009; Vinatier, Bouffi et al. 2009 ; Vinatier, Mrugala et al. 2009 ).

Differentiating MSCs and ASCs produce all the components constituting ECM and represent the cells of choice for engineering articular cartilage. However, adult chondrocytes isolated from various sources like articular cartilage, nasal septum, ribs or ear cartilage (Kafienah, Jakob et al. 2002; Isogai, Kusuhara et al. 2006) produce de novo cartilage displaying the characteristics of its original tissue (Isogai, Kusuhara et al. 2006). It is therefore more appropriate to use hyaline cartilage as the preferred source of chondrocytes, and a comparison between different sources of hyaline chondrocytes (nasal, costal, and articular) has shown the superiority of costal and nasal chondrocytes in terms of quantity of cartilage formed after subcutaneous transplantation (Isogai, Kusuhara et al. 2006). One major limit related to the use of chondrocytes, is their instability in monolayer culture resulting in loss of phenotype (i.e. loss of collagen II, aggrecan and superficial zone protein = SZP) (Darling and Athanasiou 2005). Loss of the chondrocytic phenotype is accompanied by a phenotypic shift towards fibroblast like cells, which is characterized by an enhanced expression of collagen I (Schnabel, Marlovits et al. 2002). This dedifferentiation process is reversible, and dedifferentiated chondrocytes arranged in a three-dimensional (3D) lattice may retrieve their differentiated phenotype (Domm, Schunke et al. 2002; Malda, van Blitterswijk et al. 2003). This is especially true for dedifferentiated chondrocytes, having been ”reversed” some 7-10 days before assuming the fibroblast phenotype (Brinchmann et al., unpublished observations).

The use of chondrocytes from osteoarthritic (OA) cartilage has also been contemplated. However, OA chondrocytes are subject to metabolic alterations leading to a low response to inductive environmental factors (Fukui, Purple et al. 2001; Sandell and Aigner 2001). Although chondrocytes derived from OA patients seem to be less appropriate for articular cartilage repair, it has been reported that OA chondrocytes may resume a normal chondrocytic phenotype upon 3D-cultivation in vitro (Tallheden, Bengtsson et al. 2005).


3. Genes and microRNAs as determinants of bone and cartilage quality

Characteristics of transcriptomes

When tissue replacement with the aid of tissue engineering is the ultimate therapeutic goal, it is vital to understand the differentiation process from precursor cells in terms of gene expression. Hence, it is necessary to identify a transcriptome, which is reflecting the “true” osteoblast and chondrocyte phenotypes pertaining to the function and localization of such cells in the skeleton. Many excellent articles have addressed this task over the past 10 years, of which only some are mentioned here (Kulterer, Friedl et al. 2007; Grundberg, Brandstrom et al. 2008; Duggal, Fronsdal et al. 2009; Morsczeck, Schmalz et al. 2009; Sundelacruz and Kaplan 2009; Bernstein, Sticht et al. 2010; Granchi, Ochoa et al. 2010; Piek, Sleumer et al. 2010; Sun, Mauerhan et al. 2010 ; van der Zande, Walboomers et al. 2010; Herlofsen, Kuchler et al. 2011). Two transcriptomes featuring putative gene markers of osteoblasts and chondrocytes (two each), respectively, will be described in detail.

Grundberg et al. (Grundberg, Brandstrom et al. 2008) used human trabecular osteoblasts stimulated with BMP-2 and dexamethasone for 24 hours. The article refers to genes specific for trabecular bone cells (osteoblasts) and genes up-regulated after 2 and 24 hours incubation with BMP-2 and dexamethasone, as well as genes altered upon 24 hours incubation with dexamethasone alone. Top similarity pathways of cell phenotype modulation (as assessed by Ingenuity®) were the IGF-1-, Leptin-, BMP-2- and Wnt-pathways, indicating a good correlation with the bulk of literature on osteoblast differentiation (Gordeladze, Drevon et al. 2002; Komori 2006; Marie 2008; Gordeladze, Reseland et al. 2009 ). The second paper on osteoblastogenesis (Granchi, Ochoa et al. 2010) was based on incubation of human MSCs in differentiating medium with dexamethasone for 24 hours, and thereafter in mineralizing medium with dexamethasone for 7 days. The main groups for the classifications of up-regulated genes were characterized by: angiogenesis, apoptosis, bone development, cell communication, cell cycle, embryonal development, TGFβ-signalling, and Wnt-signalling. The cumulative gene lists from these reports constituted the osteoblast transcriptome (188 genes) used as osteoblast reference to evaluate osteoblast differentiation (see paragraph 11. Bone and cartilage engineering revisited).

As for the chondrocytic differentiation, it is referred to the papers of Bernstein et al. (Bernstein, Sticht et al. 2010) and Herlofsen et al. (Herlofsen, Kuchler et al. 2011). Bernstein and co-workers used chondrocytes from articular cartilage and MSCs in an intricate array of manipulations. The cells were incubated in a differentiating medium containing TGFβ3 or control medium without growth factor. Transcriptomes obtained from the cells were categorized in gene transcripts up-regulated, down-regulated or “unsteady”. The following results were obtained: Genes were classified as belonging to groups designated TGFβ-related, Wnt-pathway, glycans, actin metabolism, integrins/motility, bone development, muscle development, neuronal development, sperm development, and lipid metabolism. Comparisons with published gene ontology (GEO) datasets revealed that 1) MSC differentiation towards the chondrogenic lineage resembled MSC differentiation in mouse embryo limb buds (endochondral differentiation), and 2) an increasing confluence of proliferating MSCs will resemble the pellet situation in a timely delayed manner (transition from proliferation to differentiation). The paper by Herlofsen et al. describes MSCs differentiated to chondrocytes in alginate beads for 21 days in a standard differentiating medium with BMP-2. The following gene transcripts COL1A1, COL2A1, COL10A1, SOX5, SOX6, SOX9, ACAN, COMP, VCAN, MMP13, ALPL, RUNX2, and SOX8 were analysed by Q-PCR for verification of differentiation. However, a similarity search, where the transcripts for COL2A1 (up-regulated upon differentiation) and CXCL12 (down-regulated upon differentiation) was compared with the time-course of other genes (1072 up-regulated, 898 down-regulated). From these exercises, a joint list was compiled, consisting of 261 genes. This cumulative list of genes was used as chondrocyte reference to evaluate chondrocyte differentiation (see paragraph 11. Bone and cartilage engineering revisited).

These are just some examples of transcriptomes characterizing cell phenotypes subsequent to exposure to differentiating conditions in vitro. Since these experiments have been conducted ex vivo, it is reasonable to anticipate that the ultimate transcriptome can only be revealed if factors and/or conditions like cell sources, growth factors (adapted incubation media), scaffolds or organ printing, mechano-stimulation, gene and/or microRNA manipulations, or gene and microRNA delivery systems are all taken into account when a final “tissue engineering” process or algorithm is selected.

Spectrum of microRNAs expressed

MicroRNAs are short (20-24 nt) non-coding single-stranded RNA molecules that play an important role in regulating cellular gene expression. They function at the post-transcriptional level, by binding mRNA molecules ( Gordeladze, Djouad et al. 2009 ; Lin 2009; Beezhold, Castranova et al. 2010). MicroRNAs have been found to play important roles in mediating fundamental biological processes like proliferation and differentiation in a variety of cells within defined tissues types. Recent reports have suggested that microRNAs may play a significant role in bone and cartilage development ( Gordeladze, Djouad et al. 2009 ; Lin 2009; Sun 2010 ; Karlsen, Shahdadfar et al. 2011).

MicroRNAs suppress target gene translation by binding to the 3'-untranslated region (3'-UTR) of mRNAs, thus repressing translation and/or enhancing mRNA degradation. This requires that the 3'-UTR contains at least one specific 6-7 nt sequence which exhibits at least partial complementarity to a so-called "seed site," located within the 5'-region of the microRNA molecule (Lin 2009; Beezhold, Castranova et al. 2010).

Despite mounting evidence that miRNAs play a significant role in embryonic development and other biological processes, the function of only a handful of miRNAs has been determined thus far. And of these miRNAs, only a small subset has been implicated in cartilage and/or bone development. These are mir-140 (targeting HDAC4), mir-199a and mir-26a (both targeting SMAD1), mir-126 (targeting VCAM1 and HOXA9), mir-125b (targeting ERBB2), mir-145, and mir-146 (Lin 2009). In a recent review article, Gordeladze et. al. summarized reports on microRNA species like mir-29b (targeting HDAC4, TGFβ3, ACVR2A, CTNNBIP1, and DUSP), mir-125b (target not specified), mir-133b and mir-135a (both targeting SMAD5 and RUNX2), and mir-199b (target not specified) somehow being involved in osteochondral development and skeletogenesis ( Gordeladze, Djouad et al. 2009 ). Other microRNAs involved in TF-interactions belong to the microRNA family of mir-23a-27a-24-2 (targeting APC2, RUNX2 and SATB2) (Hassan, Gordon et al. 2010).

Other studies involving differentiation of human mesenchymal stem cells into osteocytes and chondrocytes implicated a different subset of miRNAs. Mir-638 and mir-663 were found to be up-regulated in chondrocytes, while mir-24, let-7a, let-7b, let-7c, mir-138, and mir-320 were associated with osteocyte maturation (Lakshmipathy, Love et al. 2007). We have also found ( Gordeladze et al. , unpublished observations) that the microRNA species 638 and 663 are up-regulated in chondrocytes as early as 3 days of differentiation from MSCs, but these microRNAs are also heavily up-regulated in Th-17 cells differentiated from CD4+ naive T-cells after 5 days (Yssel et al., unpublished observations). Mir-638 and mir-663 appear to have the following targets in common (JUN, FOSB, SP3, and MYC, all of which are important for osteoblastogenesis). Finally, a recent survey of the literature revealed that several microRNA species (mir-335-5p, mir-27, and mir-29) directly target molecules involved in the Wnt-signalling pathway (Kapinas, Kessler et al. 2009; Kapinas, Kessler et al. 2010; Wang and Xu 2010; Zhang, Tu et al. 2011).

MicroRNAs directly targeting specific gene markers of bone and cartilage structural ECM molecules have not been indisputably identified, however, many microRNAs have been shown to affect the steady state levels of such molecules, though probably indirectly. These microRNAs are mir-34a, mir-675, mir-21, mir-146a (COL2A1) (Yamasaki, Nakasa et al. 2009; Abouheif, Nakasa et al. 2010; Dudek, Lafont et al. 2010; Kongcharoensombat, Nakasa et al. 2010), mir-140 (COL2A1, ACAN) (Miyaki, Nakasa et al. 2009), and mir-29b (also directly targeting COL1A1, COL5A3, AND COL4A2, as evidenced by the use of 3’-UTR reporter assays) ( Li, Hassan et al. 2009 ).

Interestingly, the expression of microRNAs in osteoblasts and chondrocytes seems to be reciprocal, in the sense that the microRNA species highly expressed in chondrocytes are virtually absent in osteoblasts (and vice versa). A series of articles on microRNA expression profiles in osteoblasts grown in a variety of scaffold composite material have recently been published (Annalisa, Furio et al. 2008; Palmieri, Pezzetti et al. 2008 ; Palmieri, Pezzetti et al. 2008 ; Palmieri, Pezzetti et al. 2008 ), however, the microRNA profiles are not overtly compatible with single microRNA studies where proof-of-microRNA-binding has been shown.

The possible role of microRNAs in disease processes like rheumatoid arthritis (RA) and osteoarthritis (OA) has been addressed, and a significant up-regulation of mir-155 and miR-146a in synovial fibroblasts (RASFs) and synovial fluid derived from patients with RA have been documented (Stanczyk, Pedrioli et al. 2008; Duroux-Richard, Presumey et al. 2011). These findings unite the concepts of cell-specific microRNA signatures and microRNA-exchange between cells in the form of exosomes (Valadi, Ekstrom et al. 2007; Zomer, Vendrig et al. 2010). Lastly, predicted polymorphisms in binding sequences for mir-146 in the promoter region of the FGF2 have been found (Lei, Papasian et al. 2011), implicating microRNAs even more closely with development and treatment of disease states.


4. Gene and microRNA manipulations and selection of delivery systems

Osteoblasts and bone engineering

Biologists have identified several bioactive factors being able to induce or support bone generation, including BMPs, TGFβ, IGF-1, FGFs, LIM mineralizing protein-1 (LMP-1), VEGF and caALK2 (activin-receptor like kinase-2, mediating BMP-signalling) (for review, see (Betz 2008)). Delivery systems frequently used are viral vectors, adenoviral vectors, retroviral and lentiviral vectors, adeno-associated vectors (AAV), and non-viral vectors (mostly plasmids). Standard transfer procedures comprise electroporation, lipofection and gene-activated matrices (GAM) (Betz 2008). Other osteogenic factors of interest for gene manipulation are RUNX2, SMADs, DLX3, DLX5, AP-1, and SP7 (osterix) (Marie 2008; Gordeladze, Djouad et al. 2009 ).

Gene therapy may be based on single genes, however, more successful attempts have been made by using combinations of genes, such as BMP + VEGF, BMP2 + RUNX2, VEGF + RANKL, BMP-2 + IGF-1, and BMP-2 + SMAD8 (Gersbach, Phillips et al. 2007 ). Another strategy is to deliver the above mentioned osteoinductive growth factors or hormones in a scaffold material to render a “kick-start” in terms of osteoblast function, cell organization and bone building (Fischer, Kolk et al. 2011). Other applications of gene therapy for osteogenesis, such as for periodontal and craniofacial regeneration, have been described elsewhere (Scheller and Krebsbach 2009; Rios, Lin et al. 2011).

Chondrocytes and cartilage engineering

The concepts of gene therapy for cartilage repair have been thoroughly reviewed by Steinert et al. (Steinert, Noth et al. 2008). Approaches mentioned are stimulation of chondrogenic differentiation (using TGFβs, BMPs, WNT, SMADs, SOX9, Brachyury), stimulation of cartilage matrix synthesis and/or cell proliferation (TGFβs, BMPs, IGF-1, PDGF, type 2 Collagen minigene, COMP, GlcAT-1), inhibition of osteogenesis/hypertrophy growth factors (Noggin, Chordin, PTHrP, SMAD6,7), the use of anti-inflammatory agents (IL-1 blockage, TNFα-inhibition, MMP-inhibition), senescence inhibition, and inhibition of apoptosis (Saraf and Mikos 2006; Steinert, Noth et al. 2008). The delivery systems for chondrogenic genes show many common features to the ones described for enhancing osteoblastogenesis (see above) (Saraf and Mikos 2006; Betz 2008).


5. Choice of humoral factors for differentiation purposes

The differentiation of progenitor cells to osteoblasts or chondrocytes in vitro has been conducted in media containing differentiating factors like Calcitriol, Dexamethasone, BMP2, IGF-1, PDGF, EGF, FGFs, TGFβs, HGF, PTH/PTHrP, (Logeart-Avramoglou, Anagnostou et al. 2005; Steinert, Noth et al. 2008; Tilg, Moschen et al. 2008; Gordeladze, Reseland et al. 2009 ; Boeuf and Richter 2010; Levi and Longaker 2011; Witkowska-Zimny and Walenko 2011). The choice of such factors, either as a single remedy, or in combinations, most certainly will affect cell phenotype acquisition in different ways (Kulterer, Friedl et al. 2007; Grundberg, Brandstrom et al. 2008; Duggal, Fronsdal et al. 2009; Sundelacruz and Kaplan 2009; Bernstein, Sticht et al. 2010; Granchi, Ochoa et al. 2010; Piek, Sleumer et al. 2010; Herlofsen, Kuchler et al. 2011). Thus, the outcome of the differentiation process is not easy to predict.

In vitro differentiation normally requires fetal bovine serum (FBS), however, FBS rises a concern over infections, possible immunological reactions to xenogenic peptides and inorganic compounds (like non-human sialic acid) (Hattori, Nogami et al. 2008). Hence, the use of serum-free incubation media is warranted. It has been shown that MSCs grown in serum free-media will acquire both osteoblast and chondrocyte phenotypes when exposed to EGF and bFGF, stimulating the ERK-pathway (Solmesky, Lefler et al. 2010), and similar results have been obtained by others (Gigout, Buschmann et al. 2009; Felka, Schafer et al. 2010). Waese et al. report on a one-step successful generation of chondrocytes in a serum-free monolayer system (with the addition of TGFβ3 or BMP-4) (Waese and Stanford 2011), and several articles underscore the importance of serum source for optimal differentiation and inhibition of senescence in engineered chondrocytes (Shahdadfar, Fronsdal et al. 2005; Dahl, Duggal et al. 2008; Duggal and Brinchmann 2011).

Hence, the combination of factors inducing optimal differentiation and the selection of serum-free media to produce good chondrocytes and osteoblasts for tissue engineering purposes represents a major task to elucidate.


6. Properties of scaffold materials in bone and cartilage engineering

Trauma (including bone fractures and cartilage destruction), cancer metastases, rheumatoid arthritis and osteoarthritis represent a therapeutic challenge, which previously has been approached by implanting autologus tissues ( Gordeladze, Reseland et al. 2009 ; Torroni 2009; Giannoudis and Dinopoulos 2010; Khan, Johnson et al. 2010; Lu, Subramony et al. 2010; Takeda, Nakagawa et al. 2011). The modern approach of using scaffolds as artificial cell- and tissue-supporting material is promising and has been extensively reviewed by Sundelacruz and Kaplan (Sundelacruz and Kaplan 2009). Basicly, che choice of scaffold biomaterial and biocompatibility is vital for support of cell proliferation, differentiation, and suitability for implantation in vivo. Secondly, the geometry and architecture is important determinants of support of 3D tissue growth, control of morphology of the growing tissue, support of cell proliferation, and favourisation of cell differentiation into particular lineages. Thirdly, the porosity of the scaffold is important for the support of cell differentiation, recruitment, aggregation, and vascularisation. Furthermore, the mechanical properties, degradation rate, and biochemical stimuli are determinant of the scaffold’s ability to permit new tissue ingrowth, allow remodelling of the ECM formed, match the healing rate of the new tissue, and stimulate progenitor cells to assume a functional and stable cell phenotype. The following scaffolds have been tested in different ostechondral tissue engineering settings: PET, PLDL, PLA, PGA, PLGA, HA, TCP, and silk fibroin (porosity and pore size), HA, TCP, various synthetic polymers and co-polymers, polymer-ceramic composites (pore inter-connectivity), natural synthetic polymers, including collagen, silk, PLGA, and PCL (degradation), natural synthetic polymers, bioactive glasses and ceramic material (mechanical strength), and finally PLGA, CaP, TCP, chitosan, HA, collagen, and silk fibroin (incorporation of biochemical signalling).

In order to arrive at the very best system for tissue engineering, large experimental permutations of the above mentioned factors including cell sources, humoral factors and gene therapeutic approaches, should be performed to obtain the better cell phenotype for osteochondral tissue replacement.

However, some recent articles featuring the use of polymeric scaffold structures in osteochondral engineering deserve citation here. Hydrogels incorporating agarose, alginate, collagen, hyaluronic acid polymer and gelatine have been successfully applied to support stem cell differentiation and 3D-structuring ( Vinatier, Bouffi et al. 2009 ; Vinatier, Mrugala et al. 2009 ; Hunt and Grover 2010). MSCs embedded in fibrin hydrogel showed superior differentiation to osteoblasts compared to cells grown in monolayers, however, they did not assume a preferred phenotype after 28 days of incubation. Tiainen and co-workers have reported on an ultra-porous titanium oxide (TiO2) scaffold with high compressive strength (above 2.5 MPa at 80-90% porosities) (Tiainen, Lyngstadaas et al. 2010), satisfying criteria for mechano-stimulation and pore size favouring cell differentiation, recruitment, aggregation and vascularisation. Another report on TiO2 confirms its applicability in producing a proper bone replacement material ( Sabetrasekh, Tiainen et al. 2011 ). Rahman et al. used bioactive glass, which, despite its brittleness, showed physical characteristics favouring neo-vascularisation being necessary for the perpetuation of engineered bone, when implanted in vivo (Rahaman, Day et al. 2011). And finally, it should be mentioned that osteoblast-like cells cultured in a bone-mimicking material made of poly-L-lactide + carbon nanotubes + micro-hydroxyapatite differentiated well into proper, bone-forming osteoblasts, as ascertained by genetic profiling (van der Zande, Walboomers et al. 2010).

Sabetrasekh and co-workers showed that Hydroxylprolyl-methyl Cellulose Hydrogel (HistocareTM) ( Sabetrasekh 2011 ) supported the differentiation of MSCs and preosteoblasts and cell clusters forming an artificial tissue favouring cell-cell interactions. Duggal et al. (Duggal, Fronsdal et al. 2009) showed that MSCs exposed to high-guluronic tripeptide arginine-glycine-aspartic acid (RDG) alginate scaffolds, facilitating binding to integrin, differentiated well into chondrocytes in the absence of any growth factors. Integrins are extracellular receptors conveying mechano-stimulation to the interior of the cell (Liu, Calderwood et al. 2000; Weyts, Li et al. 2002; Kapur, Baylink et al. 2003; Gordeladze, Reseland et al. 2009 ), and the use of RDG alginate scaffolds makes the addition of growth factors less critical for chondrocyte phenotype acquisition (as shown by transcriptome analyses). Hyalouronan (HYAFF-11®) scaffolds have been shown to produce useful cells for osteochondral tissue replacement, provided that MSCs were applied instead of ASCs in the presence of TGFβ1 (Loken, Jakobsen et al. 2008; Jakobsen, Shahdadfar et al. 2010). Finally, it should be mentioned that scaffold material (e.g. polycaprolactone, PLGA/Hap/, Collagen/Hap, agarose/gelatine hydrogel, polyacryl-amide hydrogel, PLGA nanofiber, agarose gel, polyacryl-amide gel, poly(2-hydroxyetylmethylmethacrylate) micro-porous gel, and silk fibroin) made with a gradient in pore size (Sundelacruz and Kaplan 2009; Oh, Kim et al. 2010; Seidi, Ramalingam et al. 2011) is especially well suitable for interface (i.e. ligament-to-bone, tendon-to-bone and cartilage-to-bone) tissue engineering.

The concept of scaffolds/biomaterial presently extends to include biopolymers, self assembled systems, nanoparticles, carbon nanotubes and quantum dots (Williams 2009). This definition also includes micro-structured surfaces (Kolind, Dolatshahi-Pirouz et al. 2010), shown to inhibit cell proliferation and favour differentiation, as well as UV-bioimprinting of single cell surfaces (Muys, Alkaisi et al. 2006), favouring propagation of surface-cell-cell interactions, ensuring proper development of a defined cell phenotype in a 3D-structure. Application of the scaffolds principle to create a functional 3D-tissue structure can also be refined to what is called organ printing. Organ printing is a process which is scaffold free or involving hydrogels, and is defined as layer-by-layer additive robotic bio-fabrication of 3D-functional living macro-tissues and organ constructs using tissue spheroids as building blocks. These spheroids are subject to tissue fusion, constituting the final 3D-structure of living tissue (Mironov, Visconti et al. 2009). The principles consists of three steps, including a) the production of homo-cellular aggregates, b) building hetero-cellular aggregates, and finally c) the assembly of organ-mimetics containing a 3D-vascular bed (Mironov, Visconti et al. 2009; Visconti, Kasyanov et al. 2010).

Organ print design of tissues may solve some of the problems encountered in osteochondral tissue engineering, namely vascularisation of bone tissue and gradient expression of genes from the luminal space to the bone interface of chondrocytes in hyaline cartilage, due to a lack of blood-born delivery of nutritional substances and oxygen (Salim, Nacamuli et al. 2004; Gibson, Milner et al. 2008). Transient changes in oxygen tension inhibit osteogenic differentiation, as demonstrated by reduced transcription of gene classes related to angiogenesis, family of matrix proteins, HIF-1α, as well as RUNX2, osteocalcin, and COL1A1 (Salim, Nacamuli et al. 2004). As for chondrocytes, it has been shown that high O2 tension makes them shift from producing normal articular isoforms of collagen (types II, IX, and XI) to collagen types I, III, and V (Gibson, Milner et al. 2008). High O2 levels also suppress the expression of SOX9, necessary for chondrocytic differentiation and Aggrecan synthesis (Murphy and Polak 2004). Interestingly, the microRNA species mir-210 has been shown to be enhanced by HIF-1α, thus improving tissue tolerance to low O2 levels (Huang, Le et al. 2010). This is consistent with the fact that mir-210 is down-regulated in dedifferentiated human articular chondrocytes assuming a more fibroblast/stem cell like phenotype (Karlsen, Shahdadfar et al. 2011).

Hence, there exists a plethora of scaffold materials to be considered, when optimal osteochondral tissue engineering conditions are to be defined.


7. Selection of stem cell niches and/or cell co-cultures

MSCs can be obtained from various tissues (Aicher, Buhring et al. 2010). Today the main source for isolation of MSCs in mammals is the bone marrow. However, bone marrow and other sources including placenta and adipose tissue contain MSCs displaying heterogeneous cell populations. Only a restricted number of appropriate stem cell markers have been explored so far, and it seems that the expression profile of CD-molecules differ on MSCs isolated from bone marrow, trabecular bone, dental pulp, articular cartilage, synovial membrane, adipose tissue, perivascular sites, term placenta, amnionic fluid, umbilical cord and pancreas (Bartholomew, Sturgeon et al. 2002; Dean and Bishop 2003; Le Blanc, Tammik et al. 2003; Niemeyer, Krause et al. 2006; Drosse, Volkmer et al. 2008; Gordeladze, Reseland et al. 2009 ; Aicher, Buhring et al. 2010). Knowledge of the phenotypical characteristics and the functional consequences of such subsets of MSCs might allow the development of improved regimens for regenerative medicine. MSCs, which express the specific cell adhesion molecule CD146, also known as MCAM, are well suited for bone repair. MSCs expressing CD56, CD146 and/or CD271 seem to be adaptable for the regeneration of bone, cartilage and intervertebral disks (Ohishi, Chiusaroli et al. 2009; Aicher, Buhring et al. 2010).

Using two or more MSC niches may thus prove beneficial for the generation of bone tissue (Matsumoto, Kuroda et al. 2008; Grellier, Bordenave et al. 2009 ). CD34-positive, VEGF-secreting endothelial/skeletal progenitor cells have been shown to enhance the vascularisation and speed up fracture healing (Matsumoto, Kuroda et al. 2008). Such progenitor cells are normally recruited to the bone-forming site by the CXR4/SDF-1 pathway (Otsuru, Tamai et al. 2008). Grellier and co-workers have reviewed the literature as to the use of co-cultures of osteogenic and endothelial cells ( Grellier, Bordenave et al. 2009 ). They describe combinations of osteogenic cells and endothelial cells like osteoblasts, osteoprogenitor cells, umbilical vein endothelial cells, endothelial progenitor cells, saphenous vein endothelial cells, outgrowth endothelial cells, and dermal vascular endothelial cells cultured in 2D- or 3D-structures of various scaffold materials (Villars, Bordenave et al. 2000; Wenger, Stahl et al. 2004; Kaigler, Krebsbach et al. 2005; Stahl, Wu et al. 2005; Kaigler, Krebsbach et al. 2006; Clarkin, Emery et al. 2008; Guillotin, Bareille et al. 2008; Grellier, Ferreira-Tojais et al. 2009 ; Grellier, Granja et al. 2009 ) where the endothelial cells form a tubular structure surrounded by ECM-producing and mineralizing osteoblasts ( Grellier, Bordenave et al. 2009 ).

In conclusion, co-cultures of niches of MSCs and/or vascularisation of appropriate scaffolds (e.g. scaffolds supporting ”asymmetric” differentiation of tissue-generating cells) might secure a better functional and long-lasting engineered tissue.


8. Mechano-stimulation of progenitor cells during differentiation

Mechano-biology is a relatively new research field, where most of the insight related to osteochondral tissue engineering comes from embryonic skeletal development ( Nowlan, Sharpe et al. 2010 ). However, the “mechanostat” principle was launched several decades ago by Frost and colleagues (Frost 2003; Skerry 2006; Mulvihill and Prendergast 2008). Genetic lesions or immobilization (surgical or drug-induced) lead to muscle less limbs, reduced muscle fibre size/number, or non-contractile muscles, and to underdeveloped joints and bones, mostly due to a lack of mechano-stimulation (Gomez, David et al. 2007; Kahn, Shwartz et al. 2009; Nowlan, Bourdon et al. 2010 ; Nowlan, Sharpe et al. 2010 ).

Several humoral factors, growth factors and receptors/ECM-protein/anchoring proteins share important signalling pathways, thus eventually leading to osteochondral differentiation of progenitor cells, for review, see ( Gordeladze, Reseland et al. 2009 ; Kelly and Jacobs 2010; Potier, Noailly et al. 2010). Osteochondral progenitor cells may be subjected to shear stress (by fluid flow), compressive load (scaffold compression, hydrostatic pressure), or stretching (uni-, bi-, or equi-axial) leading to both proliferation and differentiation (Potier, Noailly et al. 2010). Several mechano-modulatory regimens (featuring detailed molecular mechanisms, type of mechano-stimulation, mechanical load applied, static or intermittent load, frequencies, as well as time frame during osteoprogenitor cell differentiation) using both 2D- and 3D-incubation systems, have extensively been described elsewhere (Angele, Schumann et al. 2004; Huang, Hagar et al. 2004; Woods, Wang et al. 2005; Campbell, Lee et al. 2006; Miyanishi, Trindade et al. 2006; Sumanasinghe, Bernacki et al. 2006; Mauck, Byers et al. 2007; Mc Mahon, Campbell et al. 2008; Mc Mahon, Reid et al. 2008; Thorpe, Buckley et al. 2008; Wagner, Lindsey et al. 2008; Arnsdorf, Tummala et al. 2009 ; Arnsdorf, Tummala et al. 2009 ; Gordeladze, Reseland et al. 2009 ; Haudenschild, Hsieh et al. 2009; Li, Kupcsik et al. 2010). However, the permutation of various factors enlisted above, yielding the optimal osteochondral cells for further studies in vivo, is difficult to envisage.

Cell shape, determined by the RhoA-Rho kinase = ROCK (influencing the actin cytoskeleton), has received much attention as a controller of cell development (Mc Beath, Pirone et al. 2004; Arnsdorf, Tummala et al. 2009 ; Kelly and Jacobs 2010). This has renewed the interest in scaffold material made by nanotechnology, which is able to deliver 2D- and 3D-surfaces mimicking the ultimate surface pattern of osteoblasts and chondrocytes encountered in live tissues (Muys, Alkaisi et al. 2006; Kolind, Dolatshahi-Pirouz et al. 2010).


9. Stabilization of the osteoblast and chondrocyte cell phenotypes

In order to succeed replacing tissues like bone and cartilage, it is vital that the differentiated cells, whether pre-embedded in scaffolds or not, do not develop tumours or alter phenotype within a short period after implantation. The preferable phenotype should not lose acquired features or assume new ones. However, it has been speculated that engineered osteoblasts may be subject to premature senescence, acquire “drag-over” adipocyte characteristics, lose their ECM-synthesizing and mineralizing ability, while also enhancing osteoclast-mediated resorption yielding negative bone mass through multiple remodelling cycles. Furthermore, engineered hyaline cartilage chondrocytes may possibly shift their collagen-synthesizing and non-collagenous ECM producing profile towards hypertrophic and mineralizing chondrocytes. And chondrocytes may also recruit, activate and over-stimulate osteoclasts to resorb adjoining bone structures. Finally, it should be mentioned that engineered cartilage to replace hyaline articular cartilage also will be subject to remodelling, e.g. via the IL-1 induced Syndecan4-ERK-MMP3-ADAMT5 cleavage of Aggrecan, which is up-regulated in osteoarthritic joints (Bertrand, Cromme et al. 2010). It should also be mentioned that immune cells (e.g Th-17 cells) secrete interleukins known to differentiate and activate osteoclasts from monocytes (Weitzmann and Pacifici 2007; Adamopoulos and Bowman 2008; Tilg, Moschen et al. 2008; Hanada, Hanada et al. 2010; Pacifici 2010), and that chondrocytes exposed to exosome-like structures or certain microRNA antago- or pre-mirs (e.g. antagomir-222), are detrimental to the chondrocyte phenotype ( Gordeladze et al. , unpublished observations).

Figure 1.

Osteoblast differentiating scheme. Human mesenchymal stem cells (hMSCs) were incubated for 20 days in standard differentiating medium (containing dexamethasone), subjected to mechanical loading, transfected with the pcDNA3-Runx2 containing plasmid, grown in a 3D-lattice (PLA- or HA-based scaffolds), or transfected with antagomiRNAs corresponding to mir-328 and mir-339

It is therefore suggested that gene manipulations (at least temporal transcription control) should be considered as part of a strategy to create and stabilize in vivo engineered bone or cartilage for tissue replacement. Potentially, one should consider the transient manipulations of microRNAs, since these short RNA-molecules are known to interfere with a plethora of cell specific transcriptions factors ( Gordeladze, Djouad et al. 2009 ). MicroRNAs are also targeting epigenetic factors (Roach and Aigner 2007; Dahl, Duggal et al. 2008; Haberland, Montgomery et al. 2009; Lee, Jung et al. 2011; Mc Gee-Lawrence and Westendorf 2011) like HDACs involved in the differentiation of stem cells and stabilization of various cell phenotypes ( Li, Xie et al. 2009 ; Li, Hassan et al. 2009 ; Lee, Jung et al. 2011).


10. Bone and cartilage engineering revisited

Permutation of factors influencing cell phenotypes

There are numerous reports in the literature featuring the results of manipulations of single or a few variables known to affect the result of cell engineering based on stem cell or progenitor cell differentiation towards osteoblasts or chondrocytes to be implanted to heal osteochondral tissue lesions. These factors relate to cell source(s), application of growth factors, the use of gene therapy, application of mechano-stimulation and the selection of scaffold material (Isogai, Kusuhara et al. 2006; Gordeladze, Reseland et al. 2009 ; Aicher, Buhring et al. 2010; Granchi, Ochoa et al. 2010).

To find the combination of factors rendering engineered cells functional enough to assume a “proper” phenotype, generating tissues not deviating from their original counterparts with given characteristics, represents a painstaking task. It seems insurmountable, since the number of permutations necessary to explore all possible additive or synergistic interactions are numerous. It is therefore probably a good approach to define a set of measurable end-point characteristics for osteochondral tissues to evaluate the experimental steps taken, when going from bench to patient. Osteochondral tissues represent certain geometrical and mechanical properties (Knecht, Vanwanseele et al. 2006; Gordeladze, Reseland et al. 2009 ), as well as gene expression profiles (Grundberg, Brandstrom et al. 2008; Duggal, Fronsdal et al. 2009; Granchi, Ochoa et al. 2010; Herlofsen, Kuchler et al. 2011), which may guide the selection of major combinations of treatments, as envisaged by the permutation process. To shed light on this exercise, some bioinformatics exercises have been conducted, and some selected experiments have been described.

Permutations encompassing mechano-stimulation, 3D-growth, and manipulations of genes and microRNAs

MSCs were differentiated in standard media towards osteoblasts or chondrocytes, by subjecting them to cyclical mechano-stimulation (uni-axial stretch), gene manipulations, growth in 3D-lattices, and finally to manipulations of microRNA levels. The following test battery was used: Q-PCR analyses of osteoblast and chondrocyte “specific” transcription factors (TFs) and marker genes (e.g. RUNX2, OSTERIX = SP7, VDR, RANK-L, OPG, SOX9, GLI3, FOXO3A, WNT5A, ALPL, COL1A1, OSTEOCALCIN, OSTEOPONTIN, COL2A1, COL10A1, AGGRECAN); Q-PCR of mir-326, mir-339, mir-24, and mir-149; immunohistochemistry of COL2a1 and AGGRECAN; cell staining using Alizarin Red S and Alcian Blue; mineralization (radiology and histology) in SCID mice (m. tibialis); GAG/DNA-ratio, clinical score for micropellets and alginate beads; osteoclast resorption assay (using PBMCs + RANK-L/CSF-U on dentine slices). Some of the results obtained with MSCs differentiated towards osteoblasts are referred to in Figures 1, 2, 3 and 4.

Figure 2.

Selected results of the experiment described in Figure 1. Mechanical loading was performed in monolayers using uni-axial cell diameter alteration by 1000 µE (1E = 1 micro-strain = 1/1,000,000 alteration of the cell’s diameter) for 30 min every other day. The antago-miRNAs were transfected (by lipofection) into cells in monolayers every 5 days. Expression of genes like Runx2, Collagen-1, Osteocalcin and the microRNAs 328 and 339, were performed using Q-PCR. Furhtermore, Alizarin Red staining (indicating mineralized surface) was performed at day 20, and cells being deposited (for an additional 7 days) in the tibial muscle of SCID mice were X-rayed, harvested and subjectd to histological analyses

Figure 1 indicates the manipulation of MSCs grown in: 1) osteoblast differentiating medium 2) in mono-layers, 3) exposed to mechano-stimulation, 4) subject to up/down-regulation of TFs, and 5) grown in PLA- or HA-scaffolds (cylinders), or 5) transfected with pre- or antago-microRNAs. Figure 2 features some of the results of these single manipulations, indicating that RUNX2 over-expression is superior in terms of osteoblast differentiation, however, mechano-stimulation, and suppression of mir-328 and mir-339 also give promising osteoblasts for in vivo implantation.

Figure 3 describes the manipulation of MSCs grown in chondrocyte differentiating medium 1) in mono-layers, 2) exposed to mechano-stimulation, 3) subject to up/down-regulation of TFs, 4) grown in alginate beads or micropellets, or 5) transfected with pre- or antago-microRNAs. Figure 4 summarizes selected results of these single manipulations, indicating that suppression of RUNX2 is no better than incubation in micropellets or alginate beads, or transfecting the cells with premir-24 and premir-149. All in all, manipulating the microRNA species seem to give superior results.

Figure 3.

Chondrocyte differentiation scheme. Human mesenchymal stem cells (hMSCs) were incubated for 20 days in standard differentiating medium (containing TGFβ3), subjected to mechanical loading (1000 µE), infected with anti-Runx2-shRNA (contained within a lentiviral construct), grown in a 3D-lattice (micropellet or alginate), or transfected with premiRNAs corresponding to mir-24 and mir-149

From the above experiments, a 20 day differentiating scheme was envisaged, where gene- and microRNA-manipulated MSCs, grown in standard differentiating media, were mechano-stimulated for 10 days and thereafter incubated for another 10 days in a 3D-structure (HA-scaffold for osteoblast, and alginate beads for chondrocytes). These incubation schemes are shown in Figure 5, while results of the experiments are summarized in Table 1. By combining the different manipulations, it was shown that osteoblast and chondrocyte “specific” markers were enhanced some 3-4 fold over control MSCs differentiated in mono-layers compared to 2-3 fold for single condition manipulations. To assess the influence of inflammation (using incubation media containing interleukins and TNFα) on osteoblast or chondrocyte phenotype stability and osteoclast activation, cells were exposed to IL-1β, IL-6, IL-17 and TNFα for 14 days. Osteoclasts differentiated from PBMCs for 7 days were then co-culture with the osteoblasts or chondrocytes, and resorption pit surface was assessed. It became quite clear that inflammatory cytokines were detrimental to the ostechondral cell phenotypes and microRNA profile, and they also enhanced their ability to stimulate bone resorption through activation of osteoclasts. From these experiments, it seems that one might chose transient microRNA manipulations in combination with either cell stretching or growth in scaffold/hydrogel, if a permanent gene manipulation (e.g. alteration of RUNX2- and possibly also SOX9-expression) may render the cells less prone to negative influence encountered within their new environment.

Bioinformatics networking using micro-arrays of translated RNAs and non-translated microRNAs

To elucidate the concept of permutations of variables pertaining to the differentiation of stem cells (SCs) to become preferred osteoblasts or chondrocytes for tissue replacement, we will present an interesting exercise with transcriptomes, microRNA micro-arrays and a literature survey. Based on osteoblast derived transcriptomes (Grundberg, Brandstrom et al. 2008; Granchi, Ochoa et al. 2010), featuring gene transcripts from cells in human trabecular hip bone explants, and differentiating human mesenchymal stem cells (MSCs) undergoing differentiation and mineralization phases, respectively, a combined transcriptome of 188 genes were constructed. This transcriptome was run against two microRNA micro-arrays obtained from a) human MSCs differentiated to osteoblasts within a hydroxyapatite (HA) scaffold for 28 days, and from b) human MSCs differentiated to osteoblasts in monolayers for 3 days only, using a bioinformatics program designated Mir@nt@n (Le Bechec, Portales-Casamar et al. 2011). Furthermore, a transcriptome of genes pertinent to the chondocyte phenotype, consisting of 261 genes, was compiled by Brinchmann et al. (Duggal, Fronsdal et al. 2009; Herlofsen, Kuchler et al. 2011). These MSCs, grown in PRONOVA-LVG alginate for 21 days, represented genes displaying the same time course over 21 days as did COL2A1 or CXCL12. The present transcriptome was run against two microRNA micro-arrays obtained from a) human chondrocytes embedded in hyaline cartilage and dedifferentiated for 28 days, and b) human MSCs differentiated to chondrocytes in micropellets for 3 days.

The bioinformatics procedure featuring some comprehensive examples is given in Figure 6. Twelve genes involved in WNT- and NOTCH-mediated signalling (according to KEGG’s pathways) and a set of fourteen transcription factors (TFs) known to be important for osteoblastogenesis (Komori 2006; Marie 2008; Gordeladze, Djouad et al. 2009 ) were loaded into Mir@nt@n and two small networks emerged. All the microRNAs 16, 22, 24, 93, 125b, 141, 149, 200a and 206 have been shown to be down-regulated in osteoblastic cells ( Gordeladze, Djouad et al. 2009 ; Lin 2009), which would be consistent with an up-regulation of TFs (SATB2, ETS1, and RNF11), and WNT (signalling molecule binding to FRIZZLED-LRP5/6) ( Gordeladze, Reseland et al. 2009 ). However, NOTCH3 (known to inhibit osteoblastogenesis through interactions with the canonical WNT-pathway and Runx2) ( Gordeladze, Reseland et al. 2009 ) would also be up-regulated. Interestingly, ETS1 seems to be involved in a regulatory network involving NOTCH3, RNF11, and six microRNA species, where mir-206 is reciprocally interacting with ETS1. Mir-206 is marginally down-regulated in osteoblasts, however, significant over-expression of this microRNA species in mice leads to bone loss (Inose, Ochi et al. 2009). Finally DKK2 (an inhibitor of LPR5/6) would be up-regulated, and the present prediction cannot be given a straight-forward, simple interpretation.

Figure 4.

Selected results of the experiment described in Figure 3. Mechanical loading of MSCs was performed in monolayers using uni-axial cell diameter alteration (1000 µE) for 30 min every other day. The pre-miRNAs were transfected into cells in monolayers every 5 days. Expression of genes like Sox9, Wnt5A, Collagen-2, and the microRNAs 24 and 149, were performed using Q-PCR. Furhtermore, immunohistochemistry of Collagen 2 and Aggrecan was performed, and clinical scoring (featuring GAG/DNA-ratio, immuno-histochemistry, and distance between cells in micropellets and alginate beads) were also measured

Out of seventeen putative interactions (reciprocal or not) between microRNAs known to be down-regulated in osteoblasts, eight are compatible with a direct inhibitory effect on translation, yielding 47% consistency according to the concept of microRNA-TF interactions (Zhou, Ferguson et al. 2007; Aguda, Kim et al. 2008; Hobert 2008; Do and Scholer 2009). The conclusion to be drawn from this example is that the list of marker genes and microRNAs describing the differentiation of MSCs to osteoblasts is too slim to warrant its use as a predictor of the acquisition of a proper osteoblast phenotype to be employed in bone replacement therapy. But, after all, the marker genes and microRNAs were all just picked from various, independent articles on osteoblast differentiation and from two KEGG’s pathways charts.

Figure 5.

Multipurpose differentiation scheme. The following experimental settings were selected: Human MSCs were incubated in standard differentiating medium containing dexamethasone or TGFβ3 alone, respectively, or manipulated for 10 days interfering with gene expression, microRNA levels, and cell shape, and thereafter incubated in a 3D-stucture (HA-scaffold or alginate hydrogel, respectively) for another 10 days

More interestingly, table 2 describes the results of the use of the Mir@nt@n networking algorhitm, where the applied lists of target genes are based on cells derived from healthy human bone and cartilage, and the microRNA species are retrieved from micro-arrays obtained from the tabulated experiments. The four experimental conditions summarized here (involving on average some 30 microRNA species and 225 genes per experiment) clearly indicate that cell manipulations performed in a 3D-structure, and over a prolonged time frame of 28 days, yield a preferred osteoblast or chondrocyte phenotype, since the per cent compatibility demonstrated by microRNA – target gene interactions were 76% versus 19% (osteoblasts) and 88% versus 16% (chondrocytes), respectively.

Some examples of expected regulation patterns are given underneath: MSCs differentiated into osteoblasts in a HA scaffold show an up-regulated level of mir-143. In parallel, transcripts of putative target genes like DUSP2 (inactivates the MAPK pathway used by TNFs and TGFβs), BMP1 (involved in chondrogenesis), ID1 (belongs to the TGFβ pathway involved in chondrogenesis), TNFAIP6 (TNFα-induced protein 6), and FBN1 (sequestering TGFβ within ECM) were up-regulated. The first five interactions are expected, the last one is not (giving a per cent compatibility of 83%). Furthermore, the mir-29 family (MIR-29a,b,c) of microRNAs was down-regulated, and putative target transcripts of genes like COL1A1, COL4A1, COL4A5, COL5A1, COL21A1, BMP1, ID1, TNFAIP6, and FBN1 should be up-regulated. According to their alleged function, the interaction of the 29-family of microRNAs is expected when the collagens are concerned (Eyre 2002; Almarza and Athanasiou 2004; Goldring, Tsuchimochi et al. 2006; Davies, Chang et al. 2007; Shahdadfar, Loken et al. 2008; Heinegard 2009; Van Agtmael and Bruckner-Tuderman 2010), but does not comply with the expected down-regulation of BMP1, ID1, and TNFAIP6. Cumulative compatibility score is now down to 73%. According to the literature ( Li, Hassan et al. 2009 ), mir-29b does de facto bind to the 3’-UTR of the COL1A1, COL5A3 and COL4A2. Furthermore, the mir-29 family of microRNAs has also been shown to be involved in the regulation of Wnt-signalling through a positive feed-back loop (Kapinas, Kessler et al. 2010) and via suppression of SPARC (osteonectin) (Kapinas, Kessler et al. 2009). Scrutinizing the effect of mir-376c (being down-regulated) reveals that putative targets are DLX1 and RUNX2 (important TFs ensuring osteoblastogenesis), SPP1 (involved in bio-mineral tissue development and ossification) and PAFAH1B1 (involved in cell cycle adaptation to differentiation). Now, the cumulative compatibility score is 80%. The final cumulative score for this experiment (MSCs to osteoblasts in HA scaffold for 28 days) converged towards 76%. As expected, the 3 days incubation of MSCs seeded in culture flasks in osteogenic medium yielded a compatibility score of only 18%, where some of the microRNAs being modulated in the 28 days experiments with MSCs seeded into HA scaffolds did not appear as significantly altered (e.g. mir-376c).

Table 1.

Selected results obtained in the experiment described in Figure 5 showing synergism of the single manipulations used in combination. The following parameters were analysed: effect on cell differentiation, as estimated by Q-PCR of transcription factors (TFs) and marker genes, or as mineralized/ALP positive surfaces, in vivo mineralization in SCID mice and de novo bone tissue production (histology), and proteoglycan positive surface (Alcian blue colouration). Furthermore, the impact on cell phenotype stability upon exposure to cytokines (IL-1β, IL-6, IL-17, and TNFα) in terms of osteoclast activation and microRNA stability, was determined

Chondrocytes embedded within hyaline cartilage were dedifferentiated within their native matrix for 28 days, and a micro-RNA micro-array was obtained. Running these micro-RNA species using the Mir@nt@n algorithm together with the transcriptome of 261 genes gave a compatibility score of 88% (see Table 2). The following microRNAs and putative target gene transcripts should be mentioned: Mir-143 was up-regulated upon dedifferentiation, and putatively interacts with gene transcripts like SMO (involved in hedgehog = Hh activation of Gli1/2/3-mediated chondrogenesis) (Bale 2002; Takebe, Harris et al. 2011), COL1A1 (serves as bone matrix protein), WNT10B (involved in osteoblast differentiation), ADAMTSL1 (exhibits metalloproteinase activity), and HAS3 (synthesizes hyaluran). The mir-143 mediated suppression of all the above listed genes are expected when chondrocytes are dedifferentiated. Mir-140-3p was down-regulated and coupled to the modulation of gene transcripts like KLF4 (transcription factor activated by the Wnt-pathway) (Saulnier, Puglisi et al. 2011), FOXQ1 (serves as a down-stream mediator of TGFβ1 signalling) (Feuerborn, Srivastava et al. 2011), CITED4 (serves as a co-activator of CEP/p300, TFAP2, and SMAD4 transcription factors involved in stem cell differentiation) (Braganca, Swingler et al. 2002), and PTCH4 (receptor activated by Hh, thus stimulating the SMO-GLI pathway of gene transcription) (Takebe, Harris et al. 2011).

Figure 6.

Bioinformatics-based marker gene and microRNA networking. A list consisting of marker genes taken from the canonical WNT- and the NOTCH-pathways (see KEGG’s pathways), as well as transcription factors (TFs) and microRNAs demonstrated to be involved in differentiation of osteoblasts ( Gordeladze, Djouad et al. 2009 ; Hassan, Gordon et al. 2010; Kapinas, Kessler et al. 2010) was loaded into the Mir@nt@n algorithm, searching for interaction networks. Within the complicated network obtained, two types of interactions emerged: 1) microRNAs target several gene transcripts (putatively binding to the 3’-UTR region of the subject mRNAs) (left-hand chart), and 2) microRNAs may be involved in regulatory loops with TFs (right-hand chart)

A fall in mir-140-3p is compatible with an up-regulation of the four above mentioned gene transcripts and loss of chondrocyte phenotype. So far, the compatibility score is 100%. Another microRNA up-regulated in dedifferentiated chondrocytes is mir-382, which putatively targets the transcripts of the following genes: PLCG2 (PLCγ2 activates NF-κB, AP-1, and NFATc1 induced gene expression important for osteoblastogenesis) (Chen, Wang et al. 2008; Marie 2008; Gordeladze, Reseland et al. 2009 ), DKK2 (serves as an inhibitor of the Wnt-signalling pathway), and RUNX3 (cooperates with RUNX2 to induce chondrogenesis through Hh synthesis) (Komori 2005). Mir-15b proved to be up-regulated upon dedifferentiation of the mature, hyaline cartilage-embedded chondrocytes, and putatively targets the following gene transcripts: FGF2 (involved in chondrogenesis) (Goldring, Tsuchimochi et al. 2006), CCND1, LRP6, FZD4 (Katoh 2007). All genes targeted by mir-382 and mir-15b are associated with the osteochondral phenotype and therefore, the compatibility score remains at 100%. Lastly, mir-21 and mir-495 were up-regulated upon dedifferentiation of the hyaline cartilage chondrocytes. Putative targets were SOX5 (transcription factor favouring chondrogenesis), MEF2C (necessary factor for collagen X transcription, and interacting with Dlx5/6 to enhance RUNX2 expression) (Solomon, Berube et al. 2008), and SOX6 (transcription factor favouring chondrogenesis, FGF7 (involved in chondrogenesis), CDH13 (predisposing factor along with TGFβ3, PTHR1, and PRG1 in ossification of ligaments of the spine) (Furushima, Shimo-Onoda et al. 2002), GLI3 (early transcription factor appearing during chondrogenesis) (Bale 2002; Takebe, Harris et al. 2011), respectively. This completes the random selection of microRNAs, however, at this point the compatibility score was still a staggering 100%. Subsequent to the analysis of all putative interactions, the score fell to 88%. The same exercise performed on microRNA-arrays from MSCs differentiated in micropellets for 3 days revealed a compatibility score of some 16% only, despite a similar number of microRNAs and gene transcripts significantly modulated compared to controls.

Table 2.

Computation of compatibility score between osteoblast and chondrocyte transcriptomes and microRNA profiles. Gene transcript (mRNA) and microRNA networks were generated using osteoblast mRNA fingerprints from separate experiments (published in the literature) and microRNA-arrays from own experiments (see chapter text). Percentage of predicted mRNA-microRNA interactions in accordance with expected up-and down-regulation of gene expression in osteoblasts and chondrocytes were calculated. The higher the percentage, the better the differentiation process obtained, and (theoretically) the higher probability of success when using engineered osteochondral cells for tissue replacement

In conclusion, the more in vivo like incubation conditions, the more tissue-adapted osteoblasts and chondrocytes will be obtained when performing in vitro cell engineering. This exercise does not take into considerations all possible favourable factors (like stem cell source, differentiation media, optimal scaffolds, mechano-stimulation, gene-manipulations including phenotype protection by microRNAs etc.), but it is reasonable to believe that a permutation of selected conditions will aid in arriving at osteoblasts and chondrocytes highly suitable for long-lasting tissue replacements. Finally, it should be emphasized that one must improve on the selection of genes (and microRNAs) to constitute the preferred profile of proper osteoblasts and chondrocytes for successful tissue replacements.

Of special interest are the observations that the use of microRNA manipulations seems to protect the engineered osteoblasts and chondrocytes from losing their phenotypic characteristics in an environment where inflammation still is active, as well as protecting them from over-activating osteoclasts within the space (i.e. knee joint) where they might be replacing damaged tissue.

11. Summary and future perspectives

This chapter summarizes the concept of single factor permutations in order to arrive at the optimal scheme for generating osteochondral cells for tissue replacement. To be considered is the use of trimmed osteoblast or chondrocyte transcriptomes (between 200 and 400 transcripts) obtained from clean cell populations residing within healthy bone and cartilage, along with a defined number of microRNA species (not more than 20-30) as markers and guidance for the use of a set of manipulations eventually leading to functional and stable cell phenotypes.

One scheme may consist of the following materials and factors: MSCs or ASCs exposed to a growth factor in a serum-free differentiating medium, mechano-stimulation (adapted to optimalize differentiation of osteoblasts or chondrocytes), preferably within scaffolds (designed to display a porosity gradient), transient adjustments of the levels of certain microRNA species (down-regulated in differentiating osteoblasts, up-regulated in differentiating chondrocytes).

If the disease necessitating tissue replacement can be handled/treated successfully, manipulations of the engineered cells to withstand phenotype alterations, may not be necessary. However, in the case of osteochondral replacement in joints being subject to inflammation, it may be necessary to protect the engineered cells from changing their function (e.g. stimulating osteoclastogenesis) or showing an accelerated development of senescence, by permanently modulating expression of selected genes or microRNAs.


This project was supported by EU FP6 integrated project “Genostem cells engineering for connective tissue disorders”, Norwegian Center for Stem Cell research, The National Hospital, Oslo, Norway, and The Research Council of Norway.


  1. 1. Abouheif M. M. Nakasa T. et al. 2010Silencing microRNA-34a inhibits chondrocyte apoptosis in a rat osteoarthritis model in vitro. Rheumatology 49 11 2054 2060
  2. 2. Adamopoulos I. E. Bowman E. P. 2008Immune regulation of bone loss by Th17 cells. Arthritis research & therapy 10(5): 225 EOF
  3. 3. Aguda B. D. Kim Y. et al. 2008MicroRNA regulation of a cancer network: consequences of the feedback loops involving miR-17-92, E2F, and Myc. Proc Natl Acad Sci U S A 105 50 19678 19683
  4. 4. Aicher W. K. Buhring H. J. et al. 2010Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells-Potential and pitfalls. Advanced drug delivery reviews.
  5. 5. Almarza A. J. Athanasiou K. A. 2004Design characteristics for the tissue engineering of cartilaginous tissues. Annals of biomedical engineering 32 1 2 17
  6. 6. Angele P. Schumann D. et al. 2004Cyclic, mechanical compression enhances chondrogenesis of mesenchymal progenitor cells in tissue engineering scaffolds. Biorheology 41(3-4): 335-346.
  7. 7. Annalisa P. Furio P. et al. 2008Anorganic bovine bone and a silicate-based synthetic bone activate different microRNAs. J Oral Sci 50 3 301 307
  8. 8. Arnsdorf E. J. Tummala P. et al. 2009Non-canonical Wnt signaling and N-cadherin related beta-catenin signaling play a role in mechanically induced osteogenic cell fate. PloS one 4(4): e5388.
  9. 9. Arnsdorf E. J. Tummala P. et al. 2009Mechanically induced osteogenic differentiation--the role of RhoA, ROCKII and cytoskeletal dynamics. Journal of cell science 122(Pt 4): 546-553.
  10. 10. Bale A. E. 2002Hedgehog signaling and human disease. Annual review of genomics and human genetics 3 47 65
  11. 11. Barry F. P. Murphy J. M. 2004Mesenchymal stem cells: clinical applications and biological characterization. The international journal of biochemistry & cell biology 36 4 568 584
  12. 12. Bartholomew A. Sturgeon C. et al. 2002Mesenchymal stem cells suppress lymphocyte proliferation in vitro and prolong skin graft survival in vivo. Exp Hematol 30 1 42 48
  13. 13. Beezhold K. J. Castranova V. et al. 2010Microprocessor of microRNAs: regulation and potential for therapeutic intervention. Molecular cancer 9: 134.
  14. 14. Bernstein P. Sticht C. et al. 2010Expression pattern differences between osteoarthritic chondrocytes and mesenchymal stem cells during chondrogenic differentiation. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 18 12 1596 1607
  15. 15. Bertrand J. Cromme C. et al. 2010Molecular mechanisms of cartilage remodelling in osteoarthritis. The international journal of biochemistry & cell biology 42 10 1594 1601
  16. 16. Betz V. M. Betz O. B. Harris M. B. Vrahas M. S. Evans C. H. 2008Bone tissue engineering and repair by gene therapy. Frontiers in Bioscience 13 833 841
  17. 17. Bodle J. C. Hanson A. D. et al. 2011Adipose-Derived Stem Cells in Functional Bone Tissue Engineering: Lessons from Bone Mechanobiology. Tissue engineering. Part B, Reviews.
  18. 18. Boeuf S. Richter W. 2010Chondrogenesis of mesenchymal stem cells: role of tissue source and inducing factors. Stem cell research & therapy 1(4): 31.
  19. 19. Braganca J. Swingler T. et al. 2002Human CREB-binding protein/300transactivator with ED-rich tail (CITED) 4, a new member of the CITED family, functions as a co-activator for transcription factor AP-2. The Journal of biological chemistry 277(10): 8559-8565.
  20. 20. Campbell J. J. Lee D. A. et al. 2006Dynamic compressive strain influences chondrogenic gene expression in human mesenchymal stem cells. Biorheology 43(3-4): 455-470.
  21. 21. Camussi G. Deregibus M. C. et al. 2010Exosomes/microvesicles as a mechanism of cell-to-cell communication. Kidney international 78 9 838 848
  22. 22. Chen Y. Wang X. et al. 2008Phospholipase Cgamma2 mediates RANKL-stimulated lymph node organogenesis and osteoclastogenesis. The Journal of biological chemistry 283 43 29593 29601
  23. 23. Clarkin C. E. Emery R. J. et al. 2008Evaluation of VEGF-mediated signaling in primary human cells reveals a paracrine action for VEGF in osteoblast-mediated crosstalk to endothelial cells. Journal of cellular physiology 214 2 537 544
  24. 24. Dahl J. A. Duggal S. et al. 2008Genetic and epigenetic instability of human bone marrow mesenchymal stem cells expanded in autologous serum or fetal bovine serum. The International journal of developmental biology 52 8 1033 1042
  25. 25. Darling E. M. Athanasiou K. A. 2005Rapid phenotypic changes in passaged articular chondrocyte subpopulations. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 23 2 425 432
  26. 26. Davies S. R. Chang L. W. et al. 2007Computational identification and functional validation of regulatory motifs in cartilage-expressed genes. Genome research 17 10 1438 1447
  27. 27. Dean R. M. Bishop M. R. 2003Graft-versus-host disease: emerging concepts in prevention and therapy. Curr Hematol Rep 2 4 287 294
  28. 28. Do J. T. Scholer H. R. 2009Regulatory circuits underlying pluripotency and reprogramming. Trends Pharmacol Sci 30 6 296 302
  29. 29. Domm C. Schunke M. et al. 2002Redifferentiation of dedifferentiated bovine articular chondrocytes in alginate culture under low oxygen tension. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 10 1 13 22
  30. 30. Drosse I. Volkmer E. et al. 2008Tissue engineering for bone defect healing: an update on a multi-component approach. Injury 39 Suppl 2: S9 20
  31. 31. Dudek K. A. Lafont J. E. et al. 2010Type II collagen expression is regulated by tissue-specific miR-675 in human articular chondrocytes. The Journal of biological chemistry 285 32 24381 24387
  32. 32. Duggal S. Brinchmann J. E. 2011Importance of serum source for the in vitro replicative senescence of human bone marrow derived mesenchymal stem cells. Journal of cellular physiology.
  33. 33. Duggal S. Fronsdal K. B. et al. 2009Phenotype and gene expression of human mesenchymal stem cells in alginate scaffolds. Tissue engineering. Part A 15 7 1763 1773
  34. 34. Duroux-Richard I. Presumey J. et al. 2011MicroRNAs as new player in rheumatoid arthritis. Joint, bone, spine : revue du rhumatisme 78 1 17 22
  35. 35. Eyre D. 2002Collagen of articular cartilage. Arthritis research 4 1 30 35
  36. 36. Felka T. Schafer R. et al. 2010Animal serum-free expansion and differentiation of human mesenchymal stromal cells. Cytotherapy 12 2 143 153
  37. 37. Feuerborn A. Srivastava P. K. et al. 2011The Forkhead factor FoxQ1 influences epithelial differentiation. Journal of cellular physiology 226 3 710 719
  38. 38. Fischer J. Kolk A. et al. 2011Future of local bone regeneration- Protein versus gene therapy. Journal of cranio-maxillo-facial surgery: official publication of the European Association for Cranio-Maxillo-Facial Surgery 39 1 54 64
  39. 39. Frost H. M. 2003Bone’s mechanostat: a 2003 update. Anat Rec A Discov Mol Cell Evol Biol 275 2 1081 1101
  40. 40. Fukui N. Purple C. R. et al. 2001Cell biology of osteoarthritis: the chondrocyte’s response to injury. Current rheumatology reports 3 6 496 505
  41. 41. Furushima K. Shimo-Onoda K. et al. 2002Large-scale screening for candidate genes of ossification of the posterior longitudinal ligament of the spine. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 17 1 128 137
  42. 42. Gersbach C. A. Phillips J. E. et al. 2007Genetic engineering for skeletal regenerative medicine. Annual review of biomedical engineering 9 87 119
  43. 43. Giannoudis P. V. Dinopoulos H. T. 2010Autologous bone graft: when shall we add growth factors? The Orthopedic clinics of North America 41 1 85 94table of contents.
  44. 44. Gibson J. S. Milner P. I. et al. 2008Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis. Pflugers Archiv: European journal of physiology 455 4 563 573
  45. 45. Gigout A. Buschmann M. D. et al. 2009Chondrocytes cultured in stirred suspension with serum-free medium containing pluronic-68 aggregate and proliferate while maintaining their differentiated phenotype. Tissue engineering. Part A 15 8 2237 2248
  46. 46. Goldring M. B. Goldring S. R. 2010Articular cartilage and subchondral bone in the pathogenesis of osteoarthritis. Annals of the New York Academy of Sciences 1192 230 237
  47. 47. Goldring M. B. Tsuchimochi K. et al. 2006The control of chondrogenesis. J Cell Biochem 97 1 33 44
  48. 48. Gomez C. David V. et al. 2007Absence of mechanical loading in utero influences bone mass and architecture but not innervation in Myod-Myf5-deficient mice. Journal of anatomy 210 3 259 271
  49. 49. Gordeladze J. O. Djouad F. et al. 2009Concerted stimuli regulating osteo-chondral differentiation from stem cells: phenotype acquisition regulated by microRNAs. Acta pharmacologica Sinica 30 10 1369 1384
  50. 50. Gordeladze J. O. Drevon C. A. et al. 2002Leptin stimulates human osteoblastic cell proliferation, de novo collagen synthesis, and mineralization: Impact on differentiation markers, apoptosis, and osteoclastic signaling. J Cell Biochem 85 4 825 836
  51. 51. Gordeladze J. O. Reseland J. E. et al. 2009From stem cells to bone: phenotype acquisition, stabilization, and tissue engineering in animal models. ILAR journal / National Research Council, Institute of Laboratory Animal Resources 51 1 42 61
  52. 52. Grad S. Salzmann G. M. 2009Chondrocytes- one cell type, different subpopulations : characteristics and behavior of different types of chondrocytes and implications for tissue engineering applications]. Der Orthopade 38 11 1038 1044
  53. 53. Granchi D. Ochoa G. et al. 2010Gene expression patterns related to osteogenic differentiation of bone marrow-derived mesenchymal stem cells during ex vivo expansion. Tissue engineering. Part C, Methods 16 3 511 524
  54. 54. Grellier M. Bordenave L. et al. 2009Cell-to-cell communication between osteogenic and endothelial lineages: implications for tissue engineering. Trends in biotechnology 27 10 562 571
  55. 55. Grellier M. Ferreira-Tojais N. et al. 2009Role of vascular endothelial growth factor in the communication between human osteoprogenitors and endothelial cells. Journal of cellular biochemistry 106 3 390 398
  56. 56. Grellier M. Granja P. L. et al. 2009The effect of the co-immobilization of human osteoprogenitors and endothelial cells within alginate microspheres on mineralization in a bone defect. Biomaterials 30 19 3271 3278
  57. 57. Grimshaw M. J. Mason R. M. 2001Modulation of bovine articular chondrocyte gene expression in vitro by oxygen tension. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 9 4 357 364
  58. 58. Gruber R. 2010Cell biology of osteoimmunology. Wiener medizinische Wochenschrift 160(17-18): 438-445.
  59. 59. Grundberg E. Brandstrom H. et al. 2008Systematic assessment of the human osteoblast transcriptome in resting and induced primary cells. Physiological genomics 33 3 301 311
  60. 60. Guillotin B. Bareille R. et al. 2008Interaction between human umbilical vein endothelial cells and human osteoprogenitors triggers pleiotropic effect that may support osteoblastic function. Bone 42 6 1080 1091
  61. 61. Haberland M. Montgomery R. L. et al. 2009The many roles of histone deacetylases in development and physiology: implications for disease and therapy. Nature reviews. Genetics 10 1 32 42
  62. 62. Hanada R. Hanada T. et al. 2010Physiology and pathophysiology of the RANKL/RANK system. Biological chemistry 391 12 1365 1370
  63. 63. Hassan M. Q. Gordon J. A. et al. 2010A network connecting Runx2, SATB2, and the miR-23a~27a~24-2 cluster regulates the osteoblast differentiation program. Proceedings of the National Academy of Sciences of the United States of America 107 46 19879 19884
  64. 64. Hattori H. Nogami Y. et al. 2008Expansion and characterization of adipose tissue-derived stromal cells cultured with low serum medium. Journal of biomedical materials research. Part B, Applied biomaterials 87 1 229 236
  65. 65. Haudenschild A. K. Hsieh A. H. et al. 2009Pressure and distortion regulate human mesenchymal stem cell gene expression. Annals of biomedical engineering 37 3 492 502
  66. 66. Heinegard D. 2009Proteoglycans and more--from molecules to biology. International journal of experimental pathology 90 6 575 586
  67. 67. Herlofsen S. R. Kuchler A. M. et al. 2011Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in self-gelling alginate discs reveals novel chondrogenic signature gene clusters. Tissue engineering. Part A 17(7-8): 1003-1013.
  68. 68. Hobert O. 2008Gene regulation by transcription factors and microRNAs. Science 319 5871 1785 1786
  69. 69. Huang C. Y. Hagar K. L. et al. 2004Effects of cyclic compressive loading on chondrogenesis of rabbit bone-marrow derived mesenchymal stem cells. Stem cells 22 3 313 323
  70. 70. Huang X. Le et Q. T. al 2010MiR-210--micromanager of the hypoxia pathway. Trends in molecular medicine 16 5 230 237
  71. 71. Hunt N. C. Grover L. M. 2010Cell encapsulation using biopolymer gels for regenerative medicine. Biotechnology letters 32 6 733 742
  72. 72. Inose H. Ochi H. et al. 2009A microRNA regulatory mechanism of osteoblast differentiation. Proceedings of the National Academy of Sciences of the United States of America 106 49 20794 20799
  73. 73. Isogai N. Kusuhara H. et al. 2006Comparison of different chondrocytes for use in tissue engineering of cartilage model structures. Tissue engineering 12 4 691 703
  74. 74. Jakobsen R. B. Shahdadfar A. et al. 2010Chondrogenesis in a hyaluronic acid scaffold: comparison between chondrocytes and MSC from bone marrow and adipose tissue. Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA 18 10 1407 1416
  75. 75. Javazon E. H. Beggs K. J. et al. 2004Mesenchymal stem cells: paradoxes of passaging. Experimental hematology 32 5 414 425
  76. 76. Kafienah W. Jakob M. et al. 2002Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes. Tissue engineering 8 5 817 826
  77. 77. Kahn J. Shwartz Y. et al. 2009Muscle contraction is necessary to maintain joint progenitor cell fate. Developmental cell 16 5 734 743
  78. 78. Kaigler D. Krebsbach P. H. et al. 2006Transplanted endothelial cells enhance orthotopic bone regeneration. Journal of dental research 85 7 633 637
  79. 79. Kaigler D. Krebsbach P. H. et al. 2005Endothelial cell modulation of bone marrow stromal cell osteogenic potential. The FASEB journal: official publication of the Federation of American Societies for Experimental Biology 19 6 665 667
  80. 80. Kanczler J. M. Oreffo R. O. 2008Osteogenesis and angiogenesis: the potential for engineering bone. European cells & materials 15 100 114
  81. 81. Kapinas K. Kessler C. et al. 2010miR-29 modulates Wnt signaling in human osteoblasts through a positive feedback loop. The Journal of biological chemistry 285 33 25221 25231
  82. 82. Kapinas K. Kessler C. B. et al. 2009miR-29 suppression of osteonectin in osteoblasts: regulation during differentiation and by canonical Wnt signaling. Journal of cellular biochemistry 108 1 216 224
  83. 83. Kapur S. Baylink D. J. et al. 2003Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. Bone 32 3 241 251
  84. 84. Karlsen T. A. Shahdadfar A. et al. 2011Human primary articular chondrocytes, chondroblasts-like cells, and dedifferentiated chondrocytes: differences in gene, microRNA, and protein expression and phenotype. Tissue engineering. Part C, Methods 17 2 219 227
  85. 85. Katoh M. 2007WNT signaling pathway and stem cell signaling network. Clinical cancer research : an official journal of the American Association for Cancer Research 13 14 4042 4045
  86. 86. Kelly D. J. Jacobs C. R. 2010The role of mechanical signals in regulating chondrogenesis and osteogenesis of mesenchymal stem cells. Birth defects research. Part C, Embryo today: reviews 90 1 75 85
  87. 87. Keung A. J. Healy K. E. et al. 2010Biophysics and dynamics of natural and engineered stem cell microenvironments. Wiley interdisciplinary reviews. Systems biology and medicine 2 1 49 64
  88. 88. Khan W. S. Johnson D. S. et al. 2010The potential of stem cells in the treatment of knee cartilage defects. The Knee 17 6 369 374
  89. 89. Knecht S. Vanwanseele B. et al. 2006A review on the mechanical quality of articular cartilage- implications for the diagnosis of osteoarthritis. Clinical biomechanics 21 10 999 1012
  90. 90. Kolind K. Dolatshahi-Pirouz A. et al. 2010A combinatorial screening of human fibroblast responses on micro-structured surfaces. Biomaterials 31 35 9182 9191
  91. 91. Komori T. 2005Functions of BMPs, Runx2, and osterix in the development of bone and cartilage]. Nippon rinsho. Japanese journal of clinical medicine 63 9 1671 1677
  92. 92. Komori T. 2006Regulation of osteoblast differentiation by transcription factors. J Cell Biochem 99 5 1233 1239
  93. 93. Kongcharoensombat W. Nakasa T. et al. 2010The effect of microRNA-21 on proliferation and matrix synthesis of chondrocytes embedded in atelocollagen gel. Knee surgery, sports traumatology, arthroscopy: official journal of the ESSKA 18 12 1679 1684
  94. 94. Kuhn N. Z. Tuan R. S. 2010Regulation of stemness and stem cell niche of mesenchymal stem cells: implications in tumorigenesis and metastasis. Journal of cellular physiology 222 2 268 277
  95. 95. Kulterer B. Friedl G. et al. 2007Gene expression profiling of human mesenchymal stem cells derived from bone marrow during expansion and osteoblast differentiation. BMC genomics 8: 70.
  96. 96. Kwan M. D. Slater B. J. et al. 2008Cell-based therapies for skeletal regenerative medicine. Human molecular genetics 17(R1): R93 98
  97. 97. Lakshmipathy U. Love B. et al. 2007MicroRNA expression pattern of undifferentiated and differentiated human embryonic stem cells. Stem Cells Dev 16 6 1003 1016
  98. 98. Le Bechec A. Portales-Casamar E. et al. 2011MIR@NT@N: a framework integrating transcription factors, microRNAs and their targets to identify sub-network motifs in a meta-regulation network model. BMC bioinformatics 12: 67.
  99. 99. Le Blanc K. Tammik C. et al. 2003HLA expression and immunologic properties of differentiated and undifferentiated mesenchymal stem cells. Exp Hematol 31 10 890 896
  100. 100. Lee S. Jung J. W. et al. 2011Histone deacetylase regulates high mobility group A2-targeting microRNAs in human cord blood-derived multipotent stem cell aging. Cellular and molecular life sciences : CMLS 68 2 325 336
  101. 101. Lei S. F. Papasian C. J. et al. 2011Polymorphisms in predicted miRNA binding sites and osteoporosis. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 26 1 72 78
  102. 102. Levi B. Longaker M. T. 2011Concise review: adipose-derived stromal cells for skeletal regenerative medicine. Stem cells 29 4 576 582
  103. 103. Li H. Xie H. et al. 2009A novel microRNA targeting HDAC5 regulates osteoblast differentiation in mice and contributes to primary osteoporosis in humans. The Journal of clinical investigation 119 12 3666 3677
  104. 104. Li Z. Hassan M. Q. et al. 2009Biological functions of miR-29b contribute to positive regulation of osteoblast differentiation. The Journal of biological chemistry 284 23 15676 15684
  105. 105. Li Z. Kupcsik L. et al. 2010Mechanical load modulates chondrogenesis of human mesenchymal stem cells through the TGF-beta pathway. Journal of cellular and molecular medicine 14(6A): 1338 1346
  106. 106. Lian J. B. Stein G. S. 2003Runx2/Cbfa1: a multifunctional regulator of bone formation.Curr Pharm Des 9 32 2677 2685
  107. 107. Lian J. B. Stein G. S. et al. 2006Networks and hubs for the transcriptional control of osteoblastogenesis. Reviews in endocrine & metabolic disorders 7(1-2): 1-16.
  108. 108. Lin C. J. Liu C. J. 2009MicroRNAs in Skeletogenesis. Frontiers in Bioscience 14 2757 2764
  109. 109. Liu S. Calderwood D. A. et al. 2000Integrin cytoplasmic domain-binding proteins. J Cell Sci 113 ( Pt 20): 3563-3571.
  110. 110. Logeart-Avramoglou D. Anagnostou F. et al. 2005Engineering bone: challenges and obstacles. Journal of cellular and molecular medicine 9 1 72 84
  111. 111. Loken S. Jakobsen R. B. et al. 2008Bone marrow mesenchymal stem cells in a hyaluronan scaffold for treatment of an osteochondral defect in a rabbit model. Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA 16 10 896 903
  112. 112. Lu H. H. Subramony S. D. et al. 2010Tissue engineering strategies for the regeneration of orthopedic interfaces. Annals of biomedical engineering 38 6 2142 2154
  113. 113. Malda J. van Blitterswijk C. A. et al. 2003Expansion of bovine chondrocytes on microcarriers enhances redifferentiation. Tissue engineering 9 5 939 948
  114. 114. Marie P. J. 2008Transcription factors controlling osteoblastogenesis. Arch Biochem Biophys 473 2 98 105
  115. 115. Matsumoto T. Kuroda R. et al. 2008Circulating endothelial/skeletal progenitor cells for bone regeneration and healing. Bone 43 3 434 439
  116. 116. Mauck R. L. Byers B. A. et al. 2007Regulation of cartilaginous ECM gene transcription by chondrocytes and MSCs in 3D culture in response to dynamic loading. Biomechanics and modeling in mechanobiology 6(1-2): 113-125.
  117. 117. Mc Beath R. Pirone D. M. et al. 2004Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Developmental cell 6 4 483 495
  118. 118. Mc Gee-Lawrence M. E. Westendorf J. J. 2011Histone deacetylases in skeletal development and bone mass maintenance. Gene 474(1-2): 1-11.
  119. 119. Mc Mahon L. A. Campbell V. A. et al. 2008Involvement of stretch-activated ion channels in strain-regulated glycosaminoglycan synthesis in mesenchymal stem cell-seeded 3D scaffolds. Journal of biomechanics 41 9 2055 2059
  120. 120. Mc Mahon L. A. Reid A. J. et al. 2008Regulatory effects of mechanical strain on the chondrogenic differentiation of MSCs in a collagen-GAG scaffold: experimental and computational analysis. Annals of biomedical engineering 36 2 185 194
  121. 121. Mironov V. Visconti R. P. et al. 2009Organ printing: tissue spheroids as building blocks. Biomaterials 30 12 2164 2174
  122. 122. Miyaki S. Nakasa T. et al. 2009MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses. Arthritis and rheumatism 60 9 2723 2730
  123. 123. Miyanishi K. Trindade M. C. et al. 2006Dose- and time-dependent effects of cyclic hydrostatic pressure on transforming growth factor-beta3-induced chondrogenesis by adult human mesenchymal stem cells in vitro. Tissue engineering 12 8 2253 2262
  124. 124. Monaco E. Bionaz M. et al. 2011Strategies for regeneration of the bone using porcine adult adipose-derived mesenchymal stem cells. Theriogenology 75 8 1381 1399
  125. 125. Morsczeck C. Schmalz G. et al. 2009Gene expression profiles of dental follicle cells before and after osteogenic differentiation in vitro. Clinical oral investigations 13 4 383 391
  126. 126. Mulvihill B. M. Prendergast P. J. 2008An algorithm for bone mechanoresponsiveness: implementation to study the effect of patient-specific cell mechanosensitivity on trabecular bone loss. Comput Methods Biomech Biomed Engin 11 5 443 451
  127. 127. Murphy C. L. Polak J. M. 2004Control of human articular chondrocyte differentiation by reduced oxygen tension. Journal of cellular physiology 199 3 451 459
  128. 128. Muys J. J. Alkaisi M. M. et al. 2006Cellular replication and atomic force microscope imaging using a UV-Bioimprint technique. Nanomedicine: nanotechnology, biology, and medicine 2 3 169 176
  129. 129. Niemeyer P. Krause U. et al. 2006Mesenchymal stem cell-based HLA-independent cell therapy for tissue engineering of bone and cartilage. Curr Stem Cell Res Ther 1 1 21 27
  130. 130. Nowlan N. C. Bourdon C. et al. 2010Developing bones are differentially affected by compromised skeletal muscle formation. Bone 46 5 1275 1285
  131. 131. Nowlan N. C. Sharpe J. et al. 2010Mechanobiology of embryonic skeletal development: Insights from animal models. Birth defects research. Part C, Embryo today : reviews 90 3 203 213
  132. 132. O’Sullivan J. D’Arcy S. et al. 2011Mesenchymal chondroprogenitor cell origin and therapeutic potential. Stem cell research & therapy 2(1): 8.
  133. 133. Oh S. H. Kim T. H. et al. 2010Investigation of pore size effect on chondrogenic differentiation of adipose stem cells using a pore size gradient scaffold. Biomacromolecules 11 8 1948 1955
  134. 134. Ohishi M. Chiusaroli R. et al. 2009Osteoprotegerin abrogated cortical porosity and bone marrow fibrosis in a mouse model of constitutive activation of the PTH/PTHrP receptor. The American journal of pathology 174 6 2160 2171
  135. 135. Otsuru S. Tamai K. et al. 2008Circulating bone marrow-derived osteoblast progenitor cells are recruited to the bone-forming site by the CXCR4/stromal cell-derived factor-1 pathway. Stem cells 26 1 223 234
  136. 136. Otto W. R. Rao J. 2004Tomorrow’s skeleton staff: mesenchymal stem cells and the repair of bone and cartilage. Cell proliferation 37 1 97 110
  137. 137. Pacifici R. 2010The immune system and bone. Archives of biochemistry and biophysics 503 1 41 53
  138. 138. Palmieri A. Pezzetti F. et al. 2008Titanium acts on osteoblast translational process. The Journal of oral implantology 34 4 190 195
  139. 139. Palmieri A. Pezzetti F. et al. 2008Medpor regulates osteoblast’s microRNAs. Bio-medical materials and engineering 18 2 91 97
  140. 140. Palmieri A. Pezzetti F. et al. 2008PerioGlas regulates osteoblast RNA interfering. Journal of prosthodontics : official journal of the American College of Prosthodontists 17 7 522 526
  141. 141. Phillips J. E. Gersbach C. A. et al. 2007Virus-based gene therapy strategies for bone regeneration. Biomaterials 28 2 211 229
  142. 142. Piek E. Sleumer L. S. et al. 2010Osteo-transcriptomics of human mesenchymal stem cells: accelerated gene expression and osteoblast differentiation induced by vitamin D reveals c-MYC as an enhancer of BMP2-induced osteogenesis. Bone 46 3 613 627
  143. 143. Potier E. Noailly J. et al. 2010Directing bone marrow-derived stromal cell function with mechanics. Journal of biomechanics 43 5 807 817
  144. 144. Rahaman M. N. Day D. E. et al. 2011Bioactive glass in tissue engineering. Acta biomaterialia.
  145. 145. Raimondi M. T. (2006) 2006Engineered tissue as a model to study cell and tissue function from a biophysical perspective. Current drug discovery technologies 3 4 245 268
  146. 146. Rios H. F. Lin Z. et al. 2011Cell- and Gene-Based Therapeutic Strategies for Periodontal Regenerative Medicine. Journal of periodontology.
  147. 147. Roach H. I. Aigner T. 2007DNA methylation in osteoarthritic chondrocytes: a new molecular target. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 15 2 128 137
  148. 148. Sabetrasekh R. Reseland J. E. Busch C. Lyngstadaas S. P. 2011Hydroxyproly-methyl Cellulose hydrogel (HISTOCARETM) Supports 3D Expansion of Mesenchymal Cells for Tissue Engineering. Cells & Materials.
  149. 149. Sabetrasekh R. Tiainen H. et al. 2011A novel ultra-porous titanium dioxide ceramic with excellent biocompatibility. Journal of biomaterials applications 25 6 559 580
  150. 150. Salim A. Nacamuli R. P. et al. 2004Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts. The Journal of biological chemistry 279 38 40007 40016
  151. 151. Sandell L. J. Aigner T. 2001Articular cartilage and changes in arthritis. An introduction: cell biology of osteoarthritis. Arthritis research 3 2 107 113
  152. 152. Saraf A. Mikos A. G. 2006Gene delivery strategies for cartilage tissue engineering. Advanced drug delivery reviews 58 4 592 603
  153. 153. Saulnier N. Puglisi M. A. et al. 2011Gene profiling of bone marrow- and adipose tissue-derived stromal cells: a key role of Kruppel-like factor 4 in cell fate regulation. Cytotherapy 13 3 329 340
  154. 154. Scheller E. L. Krebsbach P. H. 2009Gene therapy: design and prospects for craniofacial regeneration. Journal of dental research 88 7 585 596
  155. 155. Schnabel M. Marlovits S. et al. 2002Dedifferentiation-associated changes in morphology and gene expression in primary human articular chondrocytes in cell culture. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 10 1 62 70
  156. 156. Segawa Y. Muneta T. et al. 2009Mesenchymal stem cells derived from synovium, meniscus, anterior cruciate ligament, and articular chondrocytes share similar gene expression profiles. Journal of orthopaedic research : official publication of the Orthopaedic Research Society 27 4 435 441
  157. 157. Seidi A. Ramalingam M. et al. 2011Gradient biomaterials for soft-to-hard interface tissue engineering. Acta biomaterialia 7 4 1441 1451
  158. 158. Shahdadfar A. Fronsdal K. et al. 2005In vitro expansion of human mesenchymal stem cells: choice of serum is a determinant of cell proliferation, differentiation, gene expression, and transcriptome stability. Stem cells 23 9 1357 1366
  159. 159. Shahdadfar A. Loken S. et al. 2008Persistence of collagen type II synthesis and secretion in rapidly proliferating human articular chondrocytes in vitro. Tissue engineering. Part A 14 12 1999 2007
  160. 160. Skerry T. M. 2006One mechanostat or many? Modifications of the site-specific response of bone to mechanical loading by nature and nurture. J Musculoskelet Neuronal Interact 6 2 122 127
  161. 161. Solmesky L. Lefler S. et al. 2010Serum free cultured bone marrow mesenchymal stem cells as a platform to characterize the effects of specific molecules. PloS one 5(9).
  162. 162. Solomon L. A. Berube N. G. et al. 2008Transcriptional regulators of chondrocyte hypertrophy. Birth defects research. Part C, Embryo today : reviews 84 2 123 130
  163. 163. Stahl A. Wu X. et al. 2005Endothelial progenitor cell sprouting in spheroid cultures is resistant to inhibition by osteoblasts: a model for bone replacement grafts. FEBS letters 579 24 5338 5342
  164. 164. Stanczyk J. Pedrioli D. M. et al. 2008Altered expression of MicroRNA in synovial fibroblasts and synovial tissue in rheumatoid arthritis. Arthritis and rheumatism 58 4 1001 1009
  165. 165. Steinert A. F. Noth U. et al. 2008Concepts in gene therapy for cartilage repair. Injury 39 Suppl 1: S97 113
  166. 166. Sumanasinghe R. D. Bernacki S. H. et al. 2006Osteogenic differentiation of human mesenchymal stem cells in collagen matrices: effect of uniaxial cyclic tensile strain on bone morphogenetic protein (BMP-2) mRNA expression. Tissue engineering 12 12 3459 3465
  167. 167. Sun X. Fu X. Han W. Zhao Y. Liu H. 2010Can controlled cellular reprogramming be achieved using microRNAs? Ageing Research Reviews 9 475 483
  168. 168. Sun Y. Mauerhan D. R. et al. 2010Analysis of meniscal degeneration and meniscal gene expression. BMC musculoskeletal disorders 11: 19.
  169. 169. Sundelacruz S. Kaplan D. L. 2009Stem cell- and scaffold-based tissue engineering approaches to osteochondral regenerative medicine. Seminars in cell & developmental biology 20 6 646 655
  170. 170. Takebe N. Harris P. J. et al. 2011Targeting cancer stem cells by inhibiting Wnt, Notch, and Hedgehog pathways. Nature reviews. Clinical oncology 8 2 97 106
  171. 171. Takeda H. Nakagawa T. et al. 2011Prevention and management of knee osteoarthritis and knee cartilage injury in sports. British journal of sports medicine 45 4 304 309
  172. 172. Tallheden T. Bengtsson C. et al. 2005Proliferation and differentiation potential of chondrocytes from osteoarthritic patients. Arthritis research & therapy 7(3): R560 568
  173. 173. Tare R. S. Kanczler J. et al. 2010Skeletal stem cells and bone regeneration: translational strategies from bench to clinic. Proceedings of the Institution of Mechanical Engineers. Part H, Journal of engineering in medicine 224 12 1455 1470
  174. 174. Thorpe S. D. Buckley C. T. et al. 2008Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells. Biochemical and biophysical research communications 377 2 458 462
  175. 175. Tiainen H. Lyngstadaas S. P. et al. 2010Ultra-porous titanium oxide scaffold with high compressive strength. Journal of materials science. Materials in medicine 21 10 2783 2792
  176. 176. Tilg H. Moschen A. R. et al. 2008Gut, inflammation and osteoporosis: basic and clinical concepts. Gut 57 5 684 694
  177. 177. Torroni A. 2009Engineered bone grafts and bone flaps for maxillofacial defects: state of the art. Journal of oral and maxillofacial surgery : official journal of the American Association of Oral and Maxillofacial Surgeons 67 5 1121 1127
  178. 178. Trouvin A. P. Goeb V. 2010Receptor activator of nuclear factor-kappaB ligand and osteoprotegerin: maintaining the balance to prevent bone loss. Clinical interventions in aging 5 345 354
  179. 179. Valadi H. Ekstrom K. et al. 2007Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nature cell biology 9 6 654 659
  180. 180. Van Agtmael T. Bruckner-Tuderman L. 2010Basement membranes and human disease. Cell and tissue research 339 1 167 188
  181. 181. van der Kraan P. M. Davidson E. N. et al. 2010Bone morphogenetic proteins and articular cartilage: To serve and protect or a wolf in sheep clothing’s? Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 18 6 735 741
  182. 182. van der Zande M. Walboomers X. F. et al. 2010Genetic profiling of osteoblast-like cells cultured on a novel bone reconstructive material, consisting of poly-L-lactide, carbon nanotubes and microhydroxyapatite, in the presence of bone morphogenetic protein-2. Acta biomaterialia 6 11 4352 4360
  183. 183. van Osch G. J. Brittberg M. et al. 2009Cartilage repair: past and future--lessons for regenerative medicine. Journal of cellular and molecular medicine 13 5 792 810
  184. 184. Villars F. Bordenave L. et al. 2000Effect of human endothelial cells on human bone marrow stromal cell phenotype: role of VEGF? Journal of cellular biochemistry 79 4 672 685
  185. 185. Vinatier C. Bouffi C. et al. 2009Cartilage tissue engineering: towards a biomaterial-assisted mesenchymal stem cell therapy. Current stem cell research & therapy 4 4 318 329
  186. 186. Vinatier C. Mrugala D. et al. 2009Cartilage engineering: a crucial combination of cells, biomaterials and biofactors. Trends Biotechnol 27 5 307 314
  187. 187. Visconti R. P. Kasyanov V. et al. 2010Towards organ printing: engineering an intra-organ branched vascular tree. Expert opinion on biological therapy 10 3 409 420
  188. 188. Waese E. Y. Stanford W. L. 2011One-step generation of murine embryonic stem cell-derived mesoderm progenitors and chondrocytes in a serum-free monolayer differentiation system. Stem cell research 6 1 34 49
  189. 189. Wagner D. R. Lindsey D. P. et al. 2008Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium. Annals of biomedical engineering 36 5 813 820
  190. 190. Wang T. Xu Z. 2010miR-27 promotes osteoblast differentiation by modulating Wnt signaling. Biochemical and biophysical research communications 402 2 186 189
  191. 191. Weitzmann M. N. Pacifici R. 2007T cells: unexpected players in the bone loss induced by estrogen deficiency and in basal bone homeostasis. Annals of the New York Academy of Sciences 1116 360 375
  192. 192. Wenger A. Stahl A. et al. 2004Modulation of in vitro angiogenesis in a three-dimensional spheroidal coculture model for bone tissue engineering. Tissue engineering 10(9-10): 1536-1547.
  193. 193. Weyts F. A. Li Y. S. et al. 2002ERK activation and alpha v beta 3 integrin signaling through Shc recruitment in response to mechanical stimulation in human osteoblasts. J Cell Biochem 87 1 85 92
  194. 194. Williams D. F. 2009On the nature of biomaterials. Biomaterials 30 30 5897 5909
  195. 195. Witkowska-Zimny M. Walenko K. 2011Stem cells from adipose tissue. Cellular & molecular biology letters 16 2 236 257
  196. 196. Woods A. Wang G. et al. 2005RhoA/ROCK signaling regulates Sox9 expression and actin organization during chondrogenesis. The Journal of biological chemistry 280 12 11626 11634
  197. 197. Yamasaki K. Nakasa T. et al. 2009Expression of MicroRNA-146a in osteoarthritis cartilage. Arthritis and rheumatism 60 4 1035 1041
  198. 198. Zhang J. Tu Q. et al. 2011Effects of miR-335 5p in modulating osteogenic differentiation by specifically down-regulating Wnt antagonist DKK1. Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.
  199. 199. Zhou Y. Ferguson J. et al. 2007Inter- and intra-combinatorial regulation by transcription factors and microRNAs. BMC Genomics 8: 396.
  200. 200. Zomer A. Vendrig T. et al. 2010Exosomes: Fit to deliver small RNA. Communicative & integrative biology 3 5 447 450

Written By

Jan O Gordeladze, Janne E Reseland, Tommy A Karlsen, Rune B Jakobsen, Lars Engebretsen, Ståle P Lyngstadaas, Isabelle Duroux-Richard, Christian Jorgensen and Jan E Brinchmann

Submitted: November 4th, 2010 Published: August 29th, 2011