Agents with proven anti-fibrotic effects in
With the contemporary active lifestyle and widespread professionalism in sport, the need for high-end injury therapies is growing. Conservative principles in managing various sports injuries usually do not meet the need of athletes and their coaches. In order to achieve better and faster recovery after injuries, significant effort has been made in the recent decade among researchers. Local growth factor application, targeted therapies using recombinant proteins and tissue engineering represent promising groups of future therapeutic options with promising results.
Healthy tendons and ligaments get injured either by a single application of force or by a repeated or sustained action that alters their mechanical characteristics. Genetic disorders, aging, decreased vascularity, endocrine influences, nutritional status, inactivity, immobilization, and exercise may cause tendon degeneration, thus rendering the tendon or ligament more susceptible to injury when force is applied. Hypovascularity is hypothesized to play the major role in this degeneration, both directly by causing an ischaemic environment for the fibroblast and indirectly both by contributing to the production of free radicals and by allowing for tissue hyperthermia to occur. Conservative management, such as rest, corticosteroid injection, orthotics, ultrasound, laser treatment, or shockwave treatment provide pain relief but, when they fail, surgery is required. Local growth factor application and tissue-engineering strategies, such as the development of scaffold microenvironments, responding cells, and signalling biofactors are currently generating potential areas for additional prospective investigation in tendon or ligament regeneration.
Cartilage tissue also comprises of limited intrinsic potential for healing due to the lack of blood supply and subsequent incomplete repair by local chondrocytes with inferior fibrocartilage formation. Surgical intervention is often the only option, but the repair of damaged cartilage is often less than satisfactory, and rarely restores full function or returns the tissue to its native normal state. The new concept of cartilage tissue preservation uses tissue engineering technologies, combining new biomaterials as a scaffold, applying growth factors and using stem cells and mechanical stimulation.
Skeletal muscle, on the other hand, has a great regenerative capacity; however, this process if often incomplete because of partial fibrous scar formation. Conservative therapies including cryotherapy, resting, physical therapy and pain relief medications don’t often give satisfactory results and can even be controversial. While surgery is reserved for bigger tissue defects, the need for antifibrotic agents to improve muscle repair after injury is obvious. These and platelet-derived growth factors represent the future of biological therapies for this common type of sports injury.
Novel tissue-specific therapies are mainly molecular, based on pathophysiological processes after injury. Although they seem to significantly accelerate healing and shorten the recovery time, their true goal is to achieve better and more functional repair.
2. Biological therapy for better muscle regeneration
While spontaneous muscle fibre regeneration usually occurs after muscle injury, this process can often be slow and incomplete and accompanied by fibrotic infiltration, which compromises the restoration of contractile function . Scar formation is the result of excessive wound healing leading to a poor functional outcome after trauma and surgery . Successful muscle repair after injury is important for restoring mobility and patients’ quality of life. We therefore have an important medical need for drugs that can promote or hasten muscle fibre regeneration, reduce fibrosis, and enhance muscle function .
Despite the clinical significance of muscle injuries, current treatment principles for injured skeletal muscle lack a firm scientific basis, and are based on performing RICE (Rest, Ice, Compression, Elevation) and sometimes prescribing non-steroidal anti-inflammatory drugs (NSAIDs). However, increasing evidence indicates that the administration of NSAIDs decreases regeneration and increases fibrosis by inhibiting inflammation [3, 4].
Incomplete muscle fibre regeneration and fibrotic infiltration can lead to long-term functional deficits and physical incapacitation . In recent years of muscle regeneration research, many agents have been described to have a significant antifibrotic effect in patients with heart or kidney disease and systemic sclerosis. Consequently, researchers are testing these for muscle healing, as therapeutic targets are the same. Although these agents play a life-saving role in the previously mentioned diseases, their importance for muscle injuries could be substantial and for athletes specifically, vital.
Transforming growth factor-Beta (TGF-β) and myostatin have been identified as the main factors that stimulate fibrotic differentiation. It has been shown in
|Autologous growth factors||Platelet-rich plasma (PrP)|
|Other bioactive agents||Mannose-6-phosphate (M6P)|
Angiotensin receptor blockers (ARBs)
2.1. Recombinant proteins in muscle regeneration
2.2. Platelet-rich plasma in muscle injury therapy
Among new therapeutic options for achieving more efficient healing, autologous thrombocytes have a very important place. Although there is no randomized prospective study confirming its value, platelet-rich plasma as a source of autologous growth factors is thought to be used by many sports physicians for treating muscle injuries. The use of platelet-derived preparations was prohibited by WADA until 2011 but was removed from the list after considering the lack of current evidence concerning the use of the method for the purposes of performance enhancement as current studies did not reveal a potential for performance enhancement beyond the therapeutic effect .
PrP (or platelet-rich plasma) may be defined as a volume of the plasma fraction of autologous blood having a platelet concentration above the baseline . Normal platelet counts in blood range from 150000/µL to 350000/µL. Platelet-rich plasma contains a 3 to 5-fold increase in growth factor (GF) concentrations, sometimes more [47,48]. Platelet-rich plasma can only be made from anticoagulated blood . The process begins by adding citrate to whole blood to bind the ionized calcium and inhibit the clotting cascade, followed by one or two centrifugation steps to separate red and white blood cells from platelets. When using anticoagulated PrP, activation is critical, as clotting results in the release of GF from the platelet α-granules (degranulation). PrP may be activated immediately before application, or it can occur
Platelet-rich plasma can potentially enhance healing by the delivery of various GF and cytokines from the α-granules contained in platelets. Platelets also contain subpopulations of α-granules that undergo differential release during activation, a potentially important point in understanding how PrP is activated and acts [47, 48]. Platelets contain, synthesize and release large amounts of biologically active proteins that promote tissue regeneration. Researchers have identified more than 1100 types of proteins inside platelets or on their surface [47-49]. The most commonly studied platelet proteins include platelet-derived growth factor (PDGF), transforming growth factor (TGF-β), platelet-derived epidermal growth factor (PD-EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor I and II (IGF-I, IGF-II), fibroblastic growth factor (FGF), and cytokines, including proteins such as platelet factor 4 (PF4) and CD40L. The roles of the above listed growth factors are listed in Table 2 [47, 48, 50].
|FGF||Blood vessels, smooth muscle, skin fibroblasts and other cells||Proliferative and angiogenic action Stimulates collagen production|
|VEGF||Blood vessels||Stimulates vascularisation by stimulating vascular endothelial cells|
|TGF-β||Blood vessels, skin cells, fibroblasts, monocytes||Stimulates fibroblast production Stimulates production of collagen type-I and fibronectin|
|PDGF A+B||Fibroblasts, smooth muscle cells, chondrocytes, osteoblasts, mesenchymal stem cells||One of the first growth factors to be expressed after injury|
|Stimulates other growth factor secretion Stimulates angiogenesis and macrophage activation|
|Chemotaxic and proliferative action on fibroblasts, stimulates collagen synthesis|
|PD-EGF||Blood vessel, skin cells fibroblasts and other cells||Stimulates epidermal regeneration and wound healing by stimulating keratinocytes and dermal fibroblasts Promotes cell growth, recruitment, differentiation|
|Stimulates cytokine secretion|
|IGF-I, II||Bone, blood vessel, skin, other tissue fibroblasts||Chemotactic for fibroblasts|
|Stimulates protein synthesis|
|Enhances bone formation|
|PF-4||Neutrophils, fibroblasts||Stimulates influx of neutrophils Chemotactic for fibroblasts|
|Myostatin,||Mainly function on bone/skeletal muscle adaptation and repair|
|Leukaemia inhibitory factor (LIF),|
|mechano growth factor (MGF),|
Bone morphogenetic protein (BMP)
|(a member of TGF-β superfamily)|
When treating ligament injuries with PrP, animal studies suggest that use of PDGF-BB may improve the quality of healing medial collateral ligaments, and in a similar way PrP may influence the healing of other ligaments . The effect of PrP may be dose and time-related . However, extra-articular ligaments showed better wound site filling and increased the presence of finbrinogen and GF when healing as compared to intra-articular ligaments (like ACL), but the application of PRP can improve the results after ACL injury .
To date, no major adverse effects of PrP have been noted in humans. No adverse effects were observed when PrP was infiltrated in 808 patients, mainly with osteoarthritis . The use of bovine thrombin for activation may cause a hypersensitivity reaction and is therefore avoided in modern preparation techniques . To date, there is no evidence of a systemic effect of local PrP injection or carcinogenesis. The latter may be mainly due to the short
The International Olympic Committee Consensus Statement expresses that current evidence suggests the use of PrP to be safe. They proposed that what type of PrP product is used and how it has been prepared, validated and tested should be made clear .
Suggested techniques for the application of PrP and post-injection recommendations of the International Olympic Committee Consensus Statement are :
PrP is considered to act best when placed at the site of injured tissue; therefore ultrasound guidance is advisable for accurate needle placement to the injured site.
With respect to tendon administration, there is no agreement on whether the needle should be placed inside the tendon or in the surrounding tendon sheath. In the presence of exudates around the tendon, it is suggested that it be evacuated before PrP is injected.
If PrP is administered at arthroscopy, it is suggested that the injection be administered after emptying the joint of arthroscopic fluid. In the case of open surgery, the application of PrP can be undertaken using one of the gel or semi-solid forms.
Patients should follow general recommendations after an injection with rest, ice, and limb elevation for 48 hours. Depending on the site of treatment and extent and duration of the condition, patients may follow an accelerated rehabilitation protocol under appropriate supervision.
In the XX
To date there are no randomized control studies confirming the real role of PrP in treating muscle injuries , nor was any sample in clinical studies large enough to represent relevant statistical data . However, the preclinical data seems to be promising enough for clinical studies to take place.
2.3. Other bioactive agents to improve muscle healing
3. Future perspectives of cartilage tissue repair
Damage to articular cartilage is of great clinical consequence since the cartilage tissue comprises of limited intrinsic potential for healing due to the lack of blood supply and subsequent incomplete repair by local chondrocytes with inferior fibrocartilage formation. Surgical intervention is often the only option, but the repair of damaged cartilage is often less than satisfactory, and rarely restores full function or returns the tissue to its native normal state.
Tissue engineering of articular cartilage still remains challenging due to the special structure of cartilage tissue consisting of multiphasic cellular architecture and great weight-bearing characteristics. Good knowledge and understanding of cartilage structure, its metabolism, and the process of chondrogenesis enables
3.1. Tissue engineering of articular cartilage
The field of tissue engineering uses the principles of cell biology, engineering, and medicine in order to produce such a construct that can successfully replace damaged tissue. Engineered tissue should comprise of the characteristics of the native intact tissue in terms of histological structure, morphology, function, and mechanical properties. The challenges of tissue engineering of articular cartilage include isolating and culturing cells to gain relevant and reproducible constructs with good durability
3.1.1. Biomaterials and scaffolds
Scaffolds are engineered extracellular matrices that serve as an artificial structure capable of supporting 3-dimensional tissue formation. Cells are often implanted or seeded into these scaffolds and different biomaterials are used that allow cell attachment, growth, differentiation, and regeneration of functional cartilage tissue. Scaffolds were developed with the aim to improve the biological performance of chondrocytes as well as render the surgical technique easier. In cartilage tissue engineering, scaffolds should comprise of the following characteristics; they should be biocompatible (not triggering inflammatory response and not toxic), offering temporary support to cells, mechanically strong to protect cells and withstand
In general, scaffolds are divided into natural material, synthetic polymers, and new materials. Natural materials include collagen, hyaluronic acid, fibrin glue, chitosan, agarose, and alginate. Their advantage is excellent biocompatibility since they are natural bodily constituents, thus degradation is physiological and non-toxic. On the other hand, their use includes sourcing, processing, and the risk of disease transmission. Synthetic polymers, especially PLA (poly alfa-hydroxil acid polymers) and PLGA (
The new approach represents the development of smart matrices that actively support cartilage formation and not only provide mechanical function but also allow control over cell metabolism, tissue formation, enable adjustment of the physical properties, inclusion of ECM motifs and active substances such as GF incorporated in microspheres to allow temporally and spatially controlled delivery of GF in scaffolds .
Although most of these developments seem to be promising for future clinical application, they are mainly used
3.1.2. Growth factors
Growth factors and their signaling pathways are the essential regulators of chondrogenesis during tissue engineering and thus are the prime candidates for engineering of cartilage tissue. Chondrogenesis is a multistep process that comprises of several steps: precursor cell condensation, differentiation towards chondrogenic phenotype, secretion of cartilage specific ECM components (collagen type II, aggrecan and others), chondrocytes proliferation in the area of growth plate, further differentiation towards hypertrophy, and replacement of cartilage with the bone tissue. All of the steps are regulated by different and overlapping signals (Figure 1).
In general, GFs are endogenous regulators of chondrogenesis and their logical choice of use appears to be promising to stimulate anabolic responses and the repair of articular cartilage. For example, in
The most important factors currently used in tissue engineering are the members of transforming growth factor β (TGF-β) family, Bone Morphogenetic Protein (BMP), Insulin-like growth factors (IGF), Fibroblast Growth Factors (FGF), especially FGF-2 and FGF-18, Epidermal Growth Factor (EGF) and Vascular-Endothelial Growth Factors (VEGFs). The summary of the effect of different GFs on chondrocytes/cartilage is presented in Table 4. It is becoming increasingly apparent that GFs work synergistically and simultaneously to induce and promote cartilage formation; e.g.: TGF-β1 and FGF-2  together with IGF-1 , BMP-7 and IGF-1 , TGFβ3 and BMP , etc. Based on the concept that several different GFs work in combination during cartilage repair, the use of PrP, autologous conditioned serum (ACS), and bone marrow concentrate were used in cartilage repair techniques .
|TGF-β||Transforming Growth Factor β||Stimulates synthesis of ECM Decreases catabolic activity|
|BMP-2||Bone Morphogenetic Protein - 2||Stimulates synthesis of ECM Increased ECM turnover (increased aggrecan degradation)|
|BMP-7||Bone Morphogenetic Protein - 7||Stimulates ECM synthesis Decrease cartilage degradation|
|IGF-1||Insulin-like growth factors||Stimulates ECM synthesis Decreases matrix catabolism|
|FGF-2||Fibroblast Growth Factors-2||Decreases aggrecanase activity Antagonizes PG synthesis|
|FGF-18||Fibroblast Growth Factors-18||Increases chondrocyte proliferation and stimulates ECM|
3.1.3. Mesenchymal stem cells
Mesenchymal stem cells (MSCs) are pluripotent cells found in multiple human adult tissues including bone marrow, synovial tissues, and adipose tissues. They are of great interest for scientists involved in cell therapy and tissue engineering since they have self-renewal capacity and multilineage differentiation potential. Depending on the cultivation conditions, they can differentiate into adipogenic, ostegenic or chondrogenic cells as well as form bone, cartilage, and fat. Currently, researchers are exploring the possibilities of manipulating the stem cells under laboratory conditions into mature chondrocytes that can then be integrated into scaffolds for later application [94,95]. There are many studies reporting the isolation and characterization of MSCs from adult human synovium and periosteum and providing evidence about their multipotency at the single cell level [96, 97]. It was also demonstrated that human MSCs from different tissues possess distinctive biological properties . Additionally, there is still the issue of whether MSCs are capable of forming stable hyaline-like cartilage as opposed to that formed during the process of endochondral ossification, which is later replaced with bone. The variability in biological responses of MSCs and no standardized MSC bioprocessing to obtain MSC preparations with consistent, reproducible, and quality-controlled biological potency for therapeutic applications limit the use of MSCs in clinical practice. On the other hand, the use of MSCs as chondrocyte substitutes in an ACI-equivalent procedure has become highly attractive since MCSs are easily accessible, easy to isolate and capable of expanding into culture as opposed to articular chondrocytes with limited proliferative capacity and rapid de-differentiation
Furthermore, MCSs appear to be immune privileged under certain conditions [94-97]. Altogether, these properties would allow the generation of large batches of quality controlled MCSs preparations ready for allogenic use. In addition, limitations in patient-to-patient variability would be circumvented . In animal models, MSCs have already shown significant potential for cartilage repair and novel approaches using MSCs as an alternative cell source to patient-derived chondrocytes are being tested [89, 99]. However, preclinical and clinical studies should be conducted in order to evaluate whether the implantation of MCSs results in a cartilage formation that is as durable as the one following the implantation of articular chondrocytes. Additionally, the application of MCSs can be further expanded to non-localized chronic lesions in osteoarthritis patients .
3.1.4. Mechanical load
A potential strategy in cartilage functional tissue engineering comprises of the effect of mechanical stimuli applied during
3.2. Clinical application of tissue engineering in cartilage repair
Cartilage repair has gained great interest since autologous cartilage implantation (ACI) has become an established treatment. The first line of treatment options remains microfracturing, due to low cost, arthroscopic procedure, and ease of performance.
Bone marrow stimulation techniques as well as ACI represent the cell-based approach for tissue regeneration in which the attendance of specific cells with the ability of proliferation and differentiation in desired cell phenotype plays a crucial role. In the case of bone marrow stimulating techniques, these cells are recruited from the bone marrow either by drilling or microfracturing; as such they are released from the medullar canal and subsequently form the blot clot on the side of the lesion. However, the final result is fibrous cartilage with inferior biomechanical properties compared to native hyaline cartilage. On the other hand, the histological analysis of random biopsy specimens after ACI procedure indicated the presence of type-II collagen and hyaline-like cartilage within the healing tissue.
ACI is both technically demanding and associated with a high percentage of reoperations. The modification of this cell therapy was designed to reduce complications such as periosteal hypertrophy, the need for second look arthroscopy, the development of fibrocartilage tissue with variable amount of hyaline cartilage, etc. The next generation of ACI was developed by replacing the periosteal patch with a biocompatible matrix and selecting cells of potentially improved chondrogenic potential. For example, second generation ACI uses collagen-covered autologous cultured chondrocyte implantation and in third-generation ACI, special cell carriers or cell-seeded scaffolds were created. Fourth generation cartilage repair focuses on growth factors and gene therapy, the use of stem cells and tissue engineering [109, 110]. In general, arthrotomy and a two-stage procedure are the most commonly used, but all-arthroscopic techniques and one-stage procedures (e.g. technique with minced articular cartilage) have become highly attractive treatment techniques. Additionally, pre-implantation chondrocyte phenotype manipulation has also shown excellent outcomes.
3.2.1. Second generation ACI
Second generation ACI is still a two-step procedure, but in contrast to classical ACI it involves culturing in 3-dimensional conditions, which favours the maintenance of phenotypic stability of chondrocytes. In particular, chondrocytes are cultured on the scaffold that is biocompatible, enables cellular growth, and as such represents the graft to be transplanted. These scaffolds/matrices containing the chondrocytes are implanted on the chondral lesion and attached with fibrin glue. In this manner, periosteal grafts and their suturing onto healthy cartilage are not necessary. These techniques were developed in an attempt to resolve some of the most common problems indicated by the standard ACI technique such as periosteal hypertrophy, which is a source of complaints about localized pain among some patients.
The scaffolds used in second generation ACI should comprise all of the following futures: biocompatibility (no inflammatory response), biodegradability (controlled rate of degradation), bioactivity (promote maintenance of phenotype and proliferation), and permeability (to ensure nutrition). Natural and synthetic scaffolds can be used. The concern about the synthetic scaffolds is the risk of the harmful effect of degradation products on surrounding tissue. A comparison of the first and second-generation ACI has shown rather equivalent short-term clinical outcomes, with similar complications and a similar rate of reoperation . A variety of scaffolds have been introduced, implanted either through a small arthrotomy or arthroscopically and will be presented briefly: collagen-covered ACI (CACI or ACI-C), Hyalograft C based on hyaluronic acid and membrane/matrix induced ACI (MACI) and others.
The main innovation in CACI is the use of bioresorable collagen membrane cover instead of the periosteal cover. Initial reports showed clinical improvements of this second generation ACI with fewer complications compared to classical ACI. The clinical and functional assessment after two years showed that 74% of patients had good or excellent results following CACI compared to 67% after classical ACI (ACI-P or PACI – periosteal ACI). Revision arthroscopy was required in 36.4% in the PACI group one year after surgery due to shaving for hypertrophy compared to none in the CACI group . In the systemic review of ACI procedures including 82 studies they reported that the failure rate was highest in PACI (7.7%) compared to CACI (1.5%). Similarly, the highest rate of unplanned re-operation was in the PACI group (27%) compared to CACI (5%) .
Matrix-induced ACI (MACI) was first introduced in 1998. In this technique, cells are seeded directly onto the surface of a biodegradable type I/III collagen membrane and as such overcome the shortcomings of the original periosteum-covered technique. The membrane is a bi-layer structure, smooth on one side and rough and more porous on the inner side, with incorporated cells to stimulate cartilage matrix specific molecules. It was shown that chondrocytes can adhere and maintain their phenotypic characteristics while seeded onto a type I/III collagen membrane . The procedure requires limited exposure of the joint ensuring shorter operation time and less morbidity. The rate of failure was low (0-6.3%) and mainly due to symptomatic graft hypertrophy or detachment. However, clinical, arthroscopic, and histological outcomes are comparable for CACI and MACI . Additionally, MACI was also significantly more effective after two years compared to microfracturing . Although significantly improved results after 5 years were reported, MACI still remains the cost-intensive alternative .
Hyalograft C implants autologous cells onto an esterified hyaluronic acid scaffold. It was reported that 76% of patients had no pain and 88% had no mobility problems. Additionally, 96% of patients’ treated knee was assessed to be normal by the surgeon and cartilage repair was graded arthroscopically as normal or nearly normal in 96%. The majority of second-look biopsies showed hyaline-like cartilage and a very low rate of complications were recorded . Several other studies also reported positive clinical results with Hyalograft C [118, 119]. Similarly, in two comparative studies, researchers found superiority over hyaluronic-acid based chondrocytes transplantation at five years follow up in respect to microfracture in young, active patients [120, 121].
3.2.2. Third and fourth generation ACI
Recently, further technological advances have led to a third-generation ACI, where chondrocytes are embedded into three-dimensionally constructed scaffolds (i.e. 3-dimensional environment) for cell growth . This novel approach uses: chondro-inductive or chondro-conductive matrix; autogenous or alogenous cells treated
Likewise, Neocart (Histogenics, Waltham, MA), a bioengineered tissue patch containing an autologous chondrocyte population matured in a biodegradable collagen matrix, uses bioreactor technology (hydrostatic pressure with modified flow rates and low oxygen) to stimulate ECM accumulation and suppress long-term degradation. A recent randomized study suggests that the safety of autologous cartilage tissue implantation, with the use of the NeoCart technique is similar to that of microfracture and associated with greater clinical efficacy at two years after treatment . However, there are still technical problems remaining regarding the initial fixation technique, subchondral and edge integration, long-term durability, etc. .
A number of new generation ACI methods for implanting cultured autologous chondrocytes in a biodegradable matrix are currently in development or testing. These include Chondroselect (characterized chondrocyte implantation, TiGenex, Phase III trial), BioCart II (ProChon Biotech, Phase II trial), Cartilix (polymer hydrogel, Cartilix), MACI® (matrix-induced ACI, Verigen, available outside of the U.S.), Cartipatch (solid scaffold with an agarose-alginate matrix, TBF Tissue Engineering, Phase III trial), NeoCart (ACI with a 3-dimensional chondromatrix, Histogenics, Phase II trial) and Hyalograft C (ACI with a hyaluronic acid-based scaffold, Fidia Advanced Polymers). Although the clinical use of these second-generation ACI products has been reported in Europe, none are approved for use in the U.S. at this time .
The future of fourth generation cartilage repair focuses on gene therapy, the use of stem cells (bone marrow, adipose, or muscle derived) and tissue engineering. MSCs are an attractive cell source due to their differentiation capacities. To expand and deliver MSCs to the site of defect, the cells should be seeded into an appropriate scaffold that is biocompatible, mechanically stable, permeable, and biodegradable. A variety of biomaterials were introduced, e.g. carbohydrate polymers (hyaluronan, agarose, alginate, PLA/PLGA) that are protein-based (collagen, fibrin, gelatin) in order to obtain homogenous distribution within a 3-dimensional matrix.
Future generations of cartilage tissue engineering will also include methods to control the genome to direct chondrogenic differentiation towards a hyaline-like pathway. In this manner, the local cellular environment can be coordinated by a tightly regulated GFs that signal molecules to regulate cellular maturation and proliferation. Additionally, with the use of gene therapy, either viral or non-viral vectors can be applied into cells, which then express chondrogenic GF. Gene transfer enables localized exposure of bioactive proteins or gene products to the site of tissue lesions. There have been numerous cDNAs cloned and used for biological stimulation of cartilage healing in terms of mitosis induction, synthesis of ECM components, induction of chondrogenesis by progenitor cells, inhibiting inflammatory response, etc. Researching involves identification and specific gene combinations that could be incorporated into vectors and delivered to target cells . Current data indicates that efficient delivery and expression of certain genes may have an effect on overall healing response in cartilage tissue and is capable of turning the repair response towards the synthesis of a more hyaline cartilage tissue . The novel approach in cartilage tissue engineering is the use of cell population certification (screening of gene markers, positive and negative factors, gene expression score - ChondroCelect), which enables prediction of whether cells are capable of making stable hyaline-like cartilage
3.2.3. One-step surgery
Recent directions in cartilage repair are moving towards the possibility of performing one-step surgery, including the use of MCS and GF, and to avoid the first surgery, harvesting cell material, and subsequent cell cultivation. Numerous studies reported that bone marrow stem cells are a useful source for restoring cartilage defects. Additionally, by the concomitant use of PRP and MCS it is possible to develop a single step procedure.
Single stage procedures can be divided into two categories: cell free implants (scaffolds) and cell-based implants (further subdivided according to the cell type utilized; auto- and allografts). One of the most common cell-free procedures is AMIC (autologous matrix-induced chondrogenesis). The technique requires a cell free implant that is “smart” enough to provide the appropriate stimuli to induce orderly and durable tissue regeneration. Moreover, it should be capable of inducing in situ cartilage formation. The AMIC procedure comprises of microfracturing combined with the implant of a porcine collagen type I/III bilayer matrix to stabilize blood clot formation.
The first reports on the AMIC technique were promising and the results were comparable to standard ACI with the advantage of a single stage technique and no donor site morbidity . In a study with the mean follow up rate of 37 months, they reported highly satisfactory results in 87% with MRI showing moderate to complete filling and normal to hyperdense signal . Another possibility is to use bone marrow concentrate (BMC) for MCS in treating cartilage defects. The technique consists of harvesting 40-60ml of bone marrow aspirate from the iliac crest, centrifugation, and the use of special enzymes to activate the BMC and produce the sticky clot material that is placed on the side of the lesion; finally the defect is covered with a collagen membrane.
An attractive option in terms of cell-based technologies is represented by minced articular cartilage procedures for repairing articular cartilage, as they are one-staged, autologous and inserted on scaffold carriers that provide chondro-milieu, mechanical protection and even distribution of the cells within the defect. The principle of the minced cartilage procedure is to obtain hyaline-like “minced” cartilage pieces supplemented with the scaffold delivery system. Minced cartilage represents the source of cells and even relatively large defects can be treated with a small amount of cells; specifically, one-tenth of the cartilage that originally covered a defect is required. The proposed advantages of this procedure over conventional treatment are the elimination of the need for in-vitro cell expansion and a second surgical procedure. Several technologies are being investigated and are in current late stage trials . The autograft cell-based procedure CAIC (Mitek, USA) is currently under phase III evaluation. During the procedure, autologous cartilage is harvested with the special shaver device, then morcellized and secured on resorbable polymer mesh with fibrin glue. DeNovo NT Graft (“Natural Tissue Graft”, Zimmer, Warsaw) is a similar application used for treatment of the cartilage lesions limited to an articular surface with intact subchondral bone. It utilizes morcelized juvenile cartilage, which is secured with the fibrin glue. As there is no use of chemicals and minimal manipulation, a DeNovo NT Graft does not require FDA approval and is currently available in the United States. Both CAIS and DeNovo NT techniques rely on chondrocytes migration out of the cartilage tissue with subsequent matrix production to fill the defect [130-132]. Early animal and preclinical models have demonstrated hyaline-like cartilage. Clinical experience is limited, with short-term studies demonstrating both procedures to be safe, feasible, and effective, with improvements in subjective patient scores, and with magnetic resonance imaging .
4. Strategies to improve ligament and tendon repair
Tendons and ligaments are avascular and hypocellular with distinct mechanical features that make them difﬁcult for currently available treatments to reach a complete functional repair of the damaged tissue. Tendon injuries, whether acute or chronic, are commonly managed either conservatively or surgically. Conservative management, such as rest, corticosteroid injection, orthotics, ultrasound, laser treatment, or shockwave provide pain relief but, when they fail, surgery is required .
Surgical repair may be indicated in acute injuries. In chronic lesions, excision of the involved area might be performed. However, repaired tendons have inferior properties when compared to healthy ones. The loss of mechanical features is mainly due to a distorted extra cellular matrix (ECM) composition and a misalignment of collagen ﬁbrils of the scar tissue . Another option is to use tendon or ligament grafts, but graft-augmentation devices and artificial prostheses have also been developed . Because current treatment is suboptimal, alternative therapies have been developed, such as the delivery of growth factors, the development of engineered scaffolds or the application of stem cells.
4.1. Grafts and graft-augmentation devices
Autografts are used widely to repair the affected tendon and prevent instability due to the damaged ligament. The most commonly used autografts include hamstring tendons (semitendinosus and gracilis) and bone-patellar ligament (middle third)-bone. Several factors are important in the selection of the graft tissue reconstruction, such as the initial mechanical properties of the graft tissue, morbidity resulting from graft harvesting, graft healing, and the initial mechanical properties of the graft fixation, .
Allografts represent an alternative option to autografts for tendon and ligament repair. Because of high cost, limited accessibility, associated risk of disease transmission and tissue rejection with the use of allografts, autografts are preferred.
Immediately after a reconstruction with autograft or allograft, the fixation site, not the graft midsubstance, is considered to be the weakest point; following that period, the process of ligamentization influences the mechanical properties of the graft, making it more vulnerable.
To prevent injuries of the graft until integration into the bone and the process of ligamentization is complete, graft augmentation devices were developed to provide immediate post-surgical protection. They share mechanical loads with the biological graft until the graft itself is capable of withstanding local tensile and compressive forces . Graft augmentation devices should be resorbable, but the rate of resorption should be limited by gradual transfer of mechanical loads to the biological graft [134, 135].
4.2. Tissue engineering
Tissue engineering (TE) combines biological materials and cells into a construct that is eventually able to replace the regenerated tissue , through the merging of three areas: scaffold microenvironment, stem cells, and signalling biofactors. The goal is to reconstruct a ligament/tendon by providing a scaffold seeded with cell-inducing neotissue formation that adequately meets the required biological and mechanical properties [136,137]. Engineering ﬁbrous tissues, such as tendons and ligaments, requires the use of ﬁbre-based scaffolds, because they should possess appropriate mechanical properties to withstand high stresses, but also high porosity and surface area to allow the seeded cells to proliferate and regenerate the tissue .
4.2.1. Stem cells and scaffolds
The purpose of TE with responding cells is to induce a regenerative response instead of scarring. Tissue engineering can be divided into two subtypes: the
Upon injury, elongated fibroblast cells resident in the tendon are activated by the inflammatory response for collagen deposition. To conduct this function, tenocytes are assisted by tendon-derived stem cells (TDSCs) .
MSCs do not differentiate spontaneously during
Adipose-derived stem cell (ASC) use for tendon regeneration and repair has recently been taken into consideration. In a recent study, the role of these stem cells in primary tendon healing has been investigated by a local autologous ASC-mixed platelet-rich plasma (PrP) application at the site of tendon injury in a control to PrP application only . The tensile strengths experimental groups were found to be significantly higher in comparison to the control group and, along with higher expression of collagen type I, FGF and VEGF levels in the experimental group, ASCs seems to enhance primary tendon healing.
It is now well accepted that seeded grafts vastly improve outcomes over un-seeded grafts. Recently, collagen matrices cultured with MSCs have appeared on the horizon for tendon repair [142-144]. The isoelectric focusing technique aligns collagen fibres to the parameters of the target tissue, adjusting the density, alignment, and strength of dense connective tissue (Gurkan: Comparison of morphology, orientation, and migration of tendon derived fibroblasts and bone marrow stromal cells on electrochemically aligned collagen constructs 2010). These matrices support a higher proliferation rate of MSCs compared to randomly oriented collagen. Currently, the versatility of synthetic polymers shows great promise in tissue engineering. Poly (1.8 octanediol-co-citrate) scaffold (POC) is a highly reproducible elastomeric material capable of being used as a synthetic scaffold to support cell growth. Instead of attaching tendinous grafts to bone via screws, the optimal approach is reconstruction using the collaboration of synthetic materials with MSCs. Paradoxically, the very complexity of the fibrocartilage interface makes it a perfect candidate for POC utilization. A scaffold with three distinct regions would allow formation of a collagenous tendon along one edge, osseous material along the other, and a middle zone representing the transition from tendon to bone. Given the capacity of MSCs to differentiate into osteogenic and tenogenic lineages, a single cell population seeded onto the scaffold could regenerate the complex fibrocartilage interface. Additionally, POC scaffolds could be crafted according to the target tendon interface, relying on Wolff's Law to govern the dynamics and load of the tendon aimed for reconstruction .
A bioreactor in TE is a device that simulates a physiological environment in order to promote cell or tissue growth In vivo. Tendons respond to mechanical forces by changing the metabolism as well as their structural and mechanical properties. Without the appropriate biomechanical stimulation, newly formed tissue will lack appropriate collagenous organization and alignment for sufﬁcient load-bearing capacity [134, 145, 146]. When subjected to mechanical stimulation In vitro, embryonic stem cells exhibited tenocyte-like morphology and positively expressed tendon-related gene markers, as well as other mechanosensory structures and molecules (cilia, integrins and myosin). In ectopic transplantation, the TE tendon under In vivo mechanical stimulus displayed more regularly aligned cells and larger collagen fibres that enabled enhanced tendon regeneration in situ, as evidenced by better histological scores and superior mechanical performance characteristics . In a recent study, rabbit flexor tendons were deprived of cells and exposed to cyclic strain in a bioreactor, in comparison to a control, which was kept unloaded in a medium for 5 days . The tendons were then implanted to bridge a zone II defect in the rabbit, followed by determination of ultimate tensile strength and elastic modulus after 4 weeks. Both were significantly improved in tendon constructs that were exposed to cyclic strain, and the histology showed an increased cellularity in the bioreactor tendons. In another study, it was showed that the material properties of human allograft tissue-engineered constructs can be enhanced by reseeding and dynamic conditioning . It was found that while conditioning duration has a significant effect on material properties, the load magnitude does not. The issue of attrition in biomechanical properties with time following cycle completion must be addressed before bioreactor preconditioning can be successfully introduced as a step in the processing of these constructs for clinical application.
4.2.3. Growth factors
Following acute tendon injury, circulation-derived cells play a crucial role in the healing processes of tissue. It was shown, that locally injected PrP is useful as an activator of circulation-derived cells for the enhancement of the initial tendon healing process . PrP also improves the mechanical properties of tendons in the early phase following acute injury, in terms of increase in the force at failure, ultimate stress, and stiffness; but the effect seems to vanish in the long-term follow up . To date, there is still a debate regarding the positive effect of PrP following acute tendon injury. There are studies that confirm the positive effect of PrP on tendon healing, since an earlier return to sports, decreased cross-sectional area of tendon, and improved earlier range of ankle motion, following Achilles tendon reconstruction was noted . It is speculated that
In chronic tendon lesions, especially tendinopathy, the use of PrP is focused on restoring normal tissue composition while avoiding further degeneration. Ultrasound-guided injections of PrP were effective in reducing pain in elbow tendinosis, medial epicondylitis  and jumper's knee . Until now, few high-quality studies on the use of autologous GF injections for the management of chronic tendinopathy showed no significant improvement compared to a control group, but in those studies, autologous blood was injected and not PrP [157-159]. Currently, there is level 3 (limited) evidence that PrP injections improve pain or function in chronic tendinopathy . More research on basic science and the clinical application of PrP needs to be undertaken before a final recommendation for PrP administration for the treatment of tendinosis can be made [47, 160].
A study performed at our department showed that the administration of PrP when reconstructing ACL with a hamstring autograft enhances early graft revascularization in the interface zone between graft and bone in the tibial tunnel; furthermore, PrP stimulates the formation of a sclerotic bony ring around the graft . Platelet-leukocyte gel, applied locally, can also improve knee stability in the first three-month period and especially in the second three-month period . Studies indicate that the delivery of PrP mimics and accelerates physiological healing and reparative tissue processes in graft healing and graft ligamentization process. Therefore, such therapy could improve knee stability and shorten the period of rehabilitation after reconstructive knee surgery. However, not all studies on humans confirm the positive effect of PrP. In one study after ACL reconstruction using patellar tendon graft with the application of PrP, researchers did not find any statistically important difference in inflammatory parameters, appearance of the graft on MRI, or clinical evaluation using validated scores . Still others did not find any differences in graft fixation after ACL reconstruction with hamstring allograft and application of PrP .
4.3. Extracorporeal shock wave therapy
Extracorporeal shock wave therapy (ESWT) is a technique used in the treatment of tendon disorders, particularly calciﬁc tendinopathy. The treatment is an extension of renal lithotripsy. It is a non-invasive modality used to stimulate healing, particularly in ligament, tendon, or bone structures. A high-energy sound wave rapidly increases pressure as it travels through the tissue, which results in cavitation that causes microtrauma. This stimulates an increase in blood flow and new blood vessel formation in the target area. Studies showed an increase in inflammatory cytokines and growth factors, as well as the regulation of tumour necrosis factor, interleukin, and bone morphogenetic protein following ESWT. Studies indicate that differentiated tenocytes are metabolically ‘‘activated’’ by ESWT and signiﬁcantly induced proliferation and production of collagen (mainly type I) compared with untreated cells [165, 166]. Not all studies were able to show a positive effect of ESWT, but this was later argued to be a possible consequence of topical anaesthetics that interfere with ESWT treatment .
Numerous other substances have been used in the treatment of tendon disorders, including sclerosants, calcium gluconate, heparin, dextrose, and aprotinin; however, more studies have to be performed to prove their efficiency . To date, no optimal treatment modalities for injured tendons or ligaments have been proposed. In fact, sheathed tendons may heal differently from those not enclosed in sheaths and the process of healing of an intra-articular ligament may differ from an extra-articular ligament. Recent studies support the idea that scaffolds can provide an alternative for tendon augmentation and that tissue engineering has an enormous therapeutic potential. In recent years studies revealed that tendon healing and regeneration may be improved by the application of several growth factors and the use of PrP expanded widely. Today, many different producers provide PrP of different composition that makes studies hard to compare. Future studies will have to explain which concentrations of PrP works the best, where it is effective and what the role of accompanying leucocytes is.
Regenerative medicine holds great promise for sports medicine with aim to develop novel therapies that will replace, repair, or promote tissue regeneration. It is an increasingly expanding area of research with hopes of providing therapeutic treatments for diseases and/or injuries that conventional medicines cannot effectively treat. Skeletal muscle has a great self-regenerative capacity, but it is unfortunately limited by fibrotic infiltration. Although none of the antifibrotic agents to improve skeletal muscle regeneration have been tested on humans to date, its clinical implications are potentially far-reaching and include not only sports-related injuries, but also diseases such as muscular dystrophies and trauma- and surgery-related injury. With emerging novel therapeutic targets this is an important area of research and presents a basis for further possibilities to study different mechanisms of action and effects drug combinations for improving muscle regeneration.
Biomaterials play an important role in directing tissue growth and may provide another tool to manipulate and control stem cell behaviour. Growth factors and therapies using mesenchymal stem cells, scaffolds, and tissue engineering using bioreactors represent promising strategies for tendon, ligament and cartilage repair. While therapies using growth factors seem to be well established in case of the first two, lack of scientific evidence still makes them questionable. In the future of cartilage repair, the modification of cellular differentiation following microfracture could be alternated with the use of exogenous growth factors and scaffolds in order to retain chongrogenic phenotype and to improve the quality of repair tissue generated in the defect. The important future prospective of cartilage repair is also focused on the quality of the bonding and integration of the newly engineered tissue to native cartilage to achieve stable healing. This holds potential for tissue-engineered strategies that would enable repairing complex cartilage lesions together with the subchondral bone and other structures. However, as with all innovations, carefully conducted studies should be carried out to access the efficiency for cartilage regeneration. Furthermore, long term prospective randomized studies are needed to confirm the encouraging preliminary results.
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