Perspective Chapter: Role of Genetics, Stem Cells in Reconstructive Surgery—Their Perspectives in Craniofacial Diseases and Syndromes

Abstract

Genetic screening, counseling, and mapping play a vital role in identification of mutant genes/chromosomes, thereby preventing the progression of the disease in craniofacial anamolies, head and neck cancer in susceptible patients. Stem cells have a wide application in treating autoimmune diseases and systemic diseases, craniofacial anomalies, head and neck cancers, esthetic and reconstructive surgery, etc. At large, surgery has been the mainstay of treatment in both disease varieties. Targeted therapies with genetic engineering and stem cell transplantation go hand in hand for improving the prognosis of these diseases to a phenomenal extent. The identification of the disease at the level of chromosomal mutation stem cell therapy in conjunction with surgery is a suitable option to obtain satisfactory results in both the disease entities. This methodical combination aids in correction of the relapse and recurrence in craniofacial anomalies as well as head and neck cancers. This chapter projects and encourages insight into the perspective approach and the importance of combining whole genome sequencing and mapping along with stem cell therapy along with the conventional treatment modalities in treatment of craniofacial deformities, head and neck neoplasms with the right timing and proper case selection to appreciate better results.

1. Introduction

Craniofacial malformations occur due to the result of an infant’s skull or facial bones fusing together very early or in an abnormal way. When the cranial bones fuse together too early, the brain gets conducive in the cranial vault and cannot expand properly due to inadequate space, which causes infant to develop craniofacial deformities and neurological problems. Craniofacial defects like cleft lip and palate, craniosynostosis/syndromes may occur because of congenital anamoly, injury or tumor.

Genetic screening and counseling imparts an immense role in the identification of the causative gene that is responsible for the mutations in craniofacial anomalies through the gene mapping and whole genomic sequencing methods which also helps in controlling the run over of the craniofacial anomalies in future generation among the affected family members. Further step ahead, genetic engineering helps to identify and correct the genetic mutations so as to plan the treatment for the affected patient on a long term basis, to give a better quality of life.

Surgery remains the treatment of choice in correction of craniofacial deformities as well as in reconstruction of craniofacial defects. Genetics and tissue engineering through stem cells have contributed in identification of gene mutations in craniofacial anomalies/syndromes and in their treatment through autologous bone grafting combined with application of stem cells engineered through in vitro and in vivo cell lineage cultures for the reconstruction of craniofacial skeleton.

2. Genetic screening and gene therapy in patients with craniofacial malformations

Genetic screening by a clinical geneticist or genetic counselor plays a pivotal role in determining the patients with isolated craniofacial anomalies and syndromes. The identification of specific syndrome is important for the overall care of the patient as it identifies the risks of other medical problems that will have to be taken into account for the overall well being of the infant as well as in the management of the craniofacial malformation/syndrome. This helps the parents and family members to understand the cause and recurrence risk with future child births. Various methods of genetic testing such as karyotype, fluorescence in situ hybridization, chromosomal microarrays and next generation sequencing can be utilized in doing the genetic screening and mapping.

Karyotype analysis finds the chromosome number and searches for deletions and duplications. The flouroscent in situ hybridisation locates specific minor deletions in genome. The chromosomal microarrays/comparative ggenomic hybridisation focusses on complete genetics at finer detail as compared with other two. Advanced technologies are based on next generation sequencing that throws lighton individual base pairs of DNA encoding proteins [1]. This contains either doing a panel of genes of a specific disorder or whole genome sequencing which is available with genetic testing companies and laboratories that offer panels of genes for specific disorders as well as whole genomic sequencing.

The clinical features of the craniofacial anomalies are peculiar as they can occur as isolated, non syndromic or as a part of Mendelian syndromes. The medical geneticist and genetic counselor determine the type of cases that which are syndromic or isolated. Studies have shown linkage of non-syndromic cleft at region of 9q21, which after subsequent fine mapping revealed the significance of forkhead box protein E1 (FOXE1). It is genetically expressed at the point of fusion between maxillary and nasal process during palate formation whose mutations resulted in cleft palate, in mice studies. The other genes are interferon regulatory factor 6(IRF 6), transforming growth factor –alfa (TGFA). GWASs have confirmed the significance of IRF6 and FOXE1, 8Q24, 10Q25 AND 17Q22 in non- syndromic cleft palate cases.

The syndromes associated with cleft lip and palate include chromosomal abnormalities like trisomy 21, 18, 13, microdeletion syndromes (22q11 deletion syndrome), autosomal dominant disorder like VanderWoude syndrome and single gene disorders. The syndromes of cleft lip alone without cleft palate result from single gene defect which account for 75% and are associated with Mendelian disorders. The cleft lip with palate and cleft palate alone are embyologically separate entity with cleft lip and palate associated with syndromes are 50% and cleft lip alone are up to 75%. The cleft palate phenotype have been identified with chromosomes like Xq21 with TBX22 gene, TBX1gene (Di George/velocardiofacial syndromes). The SATB2 gene was identified as the cause of isolated cleft palate through its role in transcriptional regulation and disruption.

On the other side of the coin, the commonest craniosynostosis syndromes are due to mutation in fibroblast growth factor receptor 2 gene (FGFR2) which alters the protein to prolong signaling such that immature embryonic cell become bone embedded cells which can promote the premature fusion of bones in the skull, hands and feet. The eight known FGFR related craniosynostosis include Crouzon syndrome (with and without acanthosis nigricans), Apert syndrome, Pfeiffer syndrome, Jakson –Weiss syndrome, Beare-Stevenson syndrome, Muenke syndrome etc. Meunke syndrome is caused by gene mutation in FGFR3 gene.

Microtia is associated with gene mutations in syndromes such as Treacher Collin’s syndrome (TCOF1, POLR 1C, POLR 1D gene), Nager syndrome (SF3B4, PRX1, PRX2 gene).

The causative gene of these craniofacial disorders/syndromes can be identified by DNA sequencing. The craniofacial malformations that are isolated, as well as that which are associated with genetic syndromes help the subject to be aware of the possible diseases and complications associated with other body systems. Majority of the craniosynostosis and syndromes can be diagnosed based on clinical findings, however, identification of genetic mutations is beneficial to the patients, family members and their next generations [2, 3].

2.1 Gene therapy in craniofacial deformities and regeneration

Conceptually, gene therapy involves insertion of new genetic material into cell to manipulate the endogenous proteins inside the cell. The methods of gene transfer are using DNA either in solution, conjugated to a biomaterial (polymers) or by viruses. In both of these, genes are transferred with in a plasmid that contains the genetic information necessary for the cell to begin making the protein product of that gene once the plasmid enters the nucleus. Viral transduction is the most effective method for gene transfer. The three main classes of viruses used for gene therapy are retroviruses, adeniviruses and adeno associated viruses. Even though the viral transduction is by far thee most effective method for gene transfer, it holds the insidious risk of insertional oncogenesis, toxic immune response, viral replication and dissemination.

Retroviruses are ideal for long term gene therapy, where in, the current human genome contains up to 5–8 of endogenous retroviral sequences that have been acquired over an evolutionary period. Adenoviruses are more suited for short term gene delivery and are used for tissue engineering that require a protein production over several weeks as they are non toxic and self limiting.

Gene therapy has been used to reprogram the fate of cells to generate induced pluripotent stem cells which change to a state of pluripotency and can differentiate to multiple different cell types. Through the insertion of specific genes involved in pluripotency of embryonic stem cells, somatic cells can be reprogrammed into cells that have self renewal and differentiation capabilities and hence are named induced pluripotent stem cells. These cells can regenerate cells from all three germ layers and can be made patient specific that overcomes the ethical issues unlike embryonic stem cells.

Adult stem cells are an excellent choice for reprogramming them into iPS stem cells due to their relative ease of isolation from different tissues, craniofacial region especially such as gingival fibroblasts, dental pulp stem cells. These cells have been successfully reprogrammed into iPS cells since these are ideal donors from the aspect of obtaining their precursor tissue from donor sites such as mandible and third molar teeth [4]. The craniofacially derived stem cells possess epigenetic memory can enhance their differentiating capability towards their tissue of origin and enhance their use for craniofacial reconstruction.

Gene therapy is used to repair and regenerate the complex tissues in craniofacial region as well as in treatment of tumor itself with genetic transfer which has made a significant progress in the last decade. This includes approval of H101 oncolytic adenovirus for treatment of head and neck cancer. Oncolytic viruses work by specifically targetting and replicating in tumor cells to result in cell death and dicrease in overall tumor size. In addition, Onco VEXR, works to regulate the immune response to the tumor by induction of antigen specific T cell responses. Genetic treatments of the tumor itself may limit the subsequent amount of reconstruction required.

In correction of craniofacial deformities, gene therapy can transform cells at the site of injury into protein synthesizing portals towards correcting them [3]. Reterovirus can be delivered directly to the desired site in which host tissue is transduced ex vivo and implanted at the site requiring tissue regeneration. Retrovirus regenerated femoral defects in rats with adenovirus transduced adipose tissue. Like plasmid DNA, viruses can be delivered on biocompatible scaffolds to generate desired protein production and tissue growth through spatial coordination of cells.

Till to date all strategies for whole tooth bioengineering have relied on the use of stem cells derived from dental pulp, periodontal ligament and/or developing tooth germ with very little emphasis on gene delivery. Further studies using recombinant adeno associated virus (AAV) is needed for bone repair due to qualities ofsuperior safety, tissue engineering and in vivo transduction. In vivo AAV mediated expression of constituently active activin receptor like kinase-2 and BMP-7 has enhanced the healing of bone defects in rodent models [5].

3. Application of stem cells in reconstruction of maxillofacial region and treatment of head and neck pathology

Stem cells have a wide application including treating variety of diseases and reconstruction of maxillofacial region, head and neck. Stem cells can be broadly classified into embryonic and adult somatic mesenchymal types. Adult stem cells are categorized into bone marrow, adipose tissue and dental sub varieties. Embryonic stem cells are categorized into somatic and pleuripotent stem cells. Both embryonic and adult stem cells can be further classified into undifferentiated, early differentiated and differentiated stem cells. The early differentiated stem cells from both the above types can be used along with the scaffold in the reconstruction of the surgical defect of maxillofacial region.

Bone marrow and adipose derived stem cells have been used along with autogenous bone grafts as scaffold in jaw reconstruction. Stem cells have been found effective in treatment of oral mucosal lesions, malignancies of the craniofacial region, along with auto immune and systemic diseases as well.

Various clinical trials are in vogue presently regarding the effect of stem cells as a treatment modality. This presentation attempted to focus insight into the application of stem cell therapy in treatment of diseases as well as reconstruction of the maxillofacial region.

Stem cells are building blocks of all organs, tissues, blood and immune system that serve as an internal repair and regeneration. Found in blood, bone marrow, muscle, adipose tissue, skin, heart, liver, placenta, amniotic fluid, membrane and sac. They lie dormant until needed to regenerate the diseased tissue. Adult humans have blood creating stem cells in bone marrow ranging between 50,000 to 2,00,000. They are activated to proliferate and differentiate into required type, upon their loss, thus maintain tissue homeostasis. Various types of stem cells are embryonic, adult, mesenchymal, tissue specific, Induced pluripotent stem cells. The adult stem cell types are hematopoetic, mesenchymal, neural, epithelial, adipose.

4. Cancer stem cells

Cancer stem cells arise from normal somatic stem cells. In the process of normal differentiation, a cell differentiates to form two cells, differentiated and primitive. A terminally differentiated cell is formed from precursor progenitor cell and finally undergoes apoptosis. CSC may originate from a normal stem cell, a normal progenitor cell or a normal differentiated cell by genetic mutation which will activate self renewal genes. During the normal differentiation process of the stem cell, instead of apoptosis, mutations occur in stem cell, progenitor cell and differentiated cell, by which they transform into respective mutated cells, there by resulting in formation of a cancer stem cell. The tumor tissue microenvironment is composed of a variety of cells, including tumor cells, cancer stem cells along with blood vessels. The cancer stem cells are rare cells found primarily in the invasive edge of tumors close to blood vessels [6].

Human cancer tissues are heterogeneous in nature and become differentiated during expansion of cancer stem cells (CSCs). CSCs initiate tumorigenesis, and are involved in tumor recurrence and metastasis. Furthermore, data show that CSCs are highly resistant to anticancer drugs. Human cancer tissues are heterogeneous in nature and become differentiated during expansion of cancer stem cells CSCs). CSCs initiate tumorigenesis, and are involved in tumor recurrence and metastasis. Furthermore, data show that CSCs are highly resistant to anticancer drugs [6].

4.1 Therapeutic targeting strategies for CSCs

Stem cells here play a dual role-in carcinogenesis and in the development of possible new cancer treatment options in future. For past so many years stem cells have been used in the replenishment of blood and immune system damage during treatment of cancer by chemotherapy or radiotherapy. Other than their use in the immuno-reconstitution, the stem cells have been reported to contribute in the tissue regeneration as they have extraordinary capacity to regenerate and differentiate. The MSCs have been used in the cell-based bone reconstruction following chemotherapy and surgery in malignancies like osteosarcoma and Ewing sarcoma [7].

Another important aspect of their use in cancer therapy is the use as delivery vehicle. Systematic delivery of drug or gene therapy has promising future but is currently limited by various factors such as immune detection, nonspecific accumulation in normal tissues and poor permeation. Stem cells can be cell based carriers that target the desired site [7, 8]. Stem cells are also used as delivery vehicles based on the hypothesis that the tumor cells send factors such as Vascular endothelial growth factor, to recruit mesenchymal stem cells from the supporting stroma of the tumor.

New techniques of targeting specific cell membrane growth factor receptors or downstream signaling pathway mutations are currently under investigation, especially in patients with metastatic tumors. One of the most promising strategies for cancer treatment is inhibiting the key self-renewal signaling pathways (e.g. Wnt, SHH, Notch signaling pathways) that are aberrantly active in CSCs, introducing novel therapeutic approaches for HNSCC. These new therapeutic techniques have a significant reduction in the CSCs, reducing its tumorigenicity, apoptotic resistance, and enhanced the sensitivity to Cancer therapy. The markers used to isolate, identify and enrich CSCs such as CD44-HYALURONIC ACID RECEPT0R, CD 24- HEAT STABLE ANTIGEN (for solid tumors), CD133, CD166, Ep CAM etc. are also ideal targets for cancer therapy.

Targeting ATP binding casette transport drugs plus other chemotherapeutic drugs, also offers a very powerful and selective strategy to eliminate CSCs. Recent therapeutic strategies exploited the interdependence of CSCs and vascular endothelial cells (perivascular niche) in head and neck cancer to decrease the rate of tumor recurrence and distant metastasis.

Compounds targeting the intrinsic and extrinsic apoptosis pathways are bicyclic cyclohexenones capable for inhibiting NF-jB signaling by inhibiting NF-jB-induced interleukin-8 (IL-8) expression, thus exerting anti-proliferative activity against lung adenocarcinoma epithelial cell line, T cell lymphoblast-like cell line, and prostate carcinoma cell line.

Nuclear factor kappa-light-chain enhancer of activated B cells (NF-jB) is a transcription factor that inhibits apoptosis by elevating the expression of survival factor. Another interesting way to manage tumor progression is inducing the terminal differentiation of CSCs to lose their self renewal property, by the means of either retinoic acids or drugs targeting tumor epigenetic changes.

4.2 Procurement and delivery of stem cells

Stem cells can be derived from the following sources like embryonic stem cells sources and adult stem cells sources. The tissue samples containing stem cells are placed under specific conditions in laboratories/stem cell banks. The extraction of these stem cells is possible due to unique receptors like Oct 4, TRA-1-60 Nanog, SSEA4, TRA-1-60 and TRA-1-81 (stem cell markers) present on the stem cell surface [7]. Tissue samples containing stem cells are placed in a sealed vial containing an appropriate media, which nourishes it during transport. The extracted stem cells are grown on a suitable scaffold medium made of biomaterials (biodegradable or non biodegradable) such as poly lactic acid, polyglycolic acid (PGA), polyethylene terepthalate, polypropylene fumarate, hydroxyapatite/tricalcium phosphate, fibrin, alginates, and collagen polytetrafluoro ethylene, fibrin sealent and certain growth factors that act as matrix during regeneration of the tissue. Stem cells are loaded in an suitable carrier called “scaffold” for transfer to desired site to close the defects or replace the organ. Scaffold can be of different shapes, pattern and biomaterials.

The sample should reach the processing storage facility before 40 hours. In the laboratory the samples were trypsinized and passaged to yield colonies of stem cells. The required cell type can be manipulated by utilizing right inductive signals and appropriate growth factors to the stem cells. Cultured stem cells should be passed through stem cell markers before it is administered to patients to know the lineage of the cell. Endotoxin test should be subjected compulsorily to the cultured stem cells to rule out any microbial contamination [9].

4.3 Mechanism of action of stem cells in defects and diseases of craniofacial region

The key role of stem cell therapy in oral mucosal lesions is primarily aimed at neoangiogenesis, tissue regeneration, increased cellularity, modulation of collagen gene expression and immunomodulation, thus making it a versatile promising treatment modality.

Stem cells are divided into embryonic and adult types. Among these the embryonic stem cells are derived by invitro fertilization, elective abortion, somatic cell nuclear transfer and cloning. The adult stem cells can be obtained from three sources such as [2] bone marrow (subdivided into hematopoetic and mesenchymal), [3] oro facial region (decidous teeth, tooth follicle, buccal mucosa, alveolar bone, periodontal ligament, dental pulp, periosteum) [4] other body tissues (skin, adipose tissue).

At present the stem cell research is focused on treatment of oral mucosal lesions such as oral mucositis, oral ulcers, pemphigus vulgaris, oral lichen planus, submucous fibrosis, oral carcinomas etc. In oral submucous fibrosis it is believed to act in removal of pathologically altered collagen and stimulation of healthy collagen through collagen gene expression and immunomodulation (b) Promoting neoangiogenesis. (c) promoting antioxidant action [7].

5. Overall potential uses of stem cells

1. Serve as repair system for the body-The pluripotent nature allows to divide into more stem cells that can be used to regenerate or repair diseased tissue and organs. Eg: mesenchymal stromal cells accelerate wound healing, by modulating immune response and promoting angiogenesis by releasing chemo cytokines and growth factors.

2. Reconstructive surgery- tissue defects, malformations, esthetics procedures.

   • For treatment of bony and soft tissue defects due to trauma, burns, non healing wounds complicated by ischemia due to diabetes.-Scar revision, skin rejuvination, hair transplantation, breast augmentation.

   • Can reduce surgical risk in elderly patients w.r.t donor site morbidity. -Increase the survival of fat graft by cell assisted lipo-transfer.-Prevent allotransplant rejection by establishing lifelong tolerance- in composite tissue allo transplantation cases in residual defects of trauma, congenital anamolies, tumor ablation etc., thus avoiding the use of immuno suppressive agents.

3. Diseases – To know how diseases occur or why certain cells develop into cancer cells.

Eg: Spinal cord injuries, osteopetrosis, Diabetes type I, Parkinson’s, Alzeimer’s, Heart and brain stroke, liver and kidney diseases, cancer and osteoarthritis, acute leukemia, CML, CLL, aplastic anemia, refractory anemia, congenital thrombocytopenia, Myelodysplastic syndrome, familial lympho histiocytosis etc. and to elicit causes of genetic defects in cells.

1. Induced pleuripotent stem cells: To grow new cells in a laboratory to replace damaged organs or tissues and body systems by genetic reprogramming (SKIN, BLOOD ETC).

2. Drugs: To test and deliver new drugs for effectiveness to the target area.

5.1 General principles

Autologous stem cell/bone marrow transplantation is healthy. Donor must be at least 18 years and above to give legal informed consent. Transplanted patients are required to live in isolation for 100 days while the new immune system establishes. Regenerative power of stem cells declines with age which can be modualted by food and life style habits. The number of stem cells needed varies with the treatment choice or the number of doses requested. The ideal number is 5–10 million/kg of receipient’s weight per transplant dose. The minimum no. is 1–2 million stem cells/kg per transplant dose [10]. The donor’s stem cells for an allogenic transplant are given to a chemotherapy/radiotherapy patient. These patients tend to have graft vs. cancer cell effect during allo- transplant. Graft failure happens when immune system rejects donor’s stem cells.

If more donor stem cells are available, second transplant or with an infusion of residual lymphocytes from the donor may be done. Donors with kidney diseases such as chronic glomerulo nephritis or polycystic kidney disease, nephrectomy patients and are over 40 years old would not able to donate stem cells.

When stem cells/bone marrow are taken from donor, they must have a similar genetic make up. Usually siblings, or a parent or unrelated person should have same genetic component. The match ratio is 1 in 4(match related donor transplant). Others are unlikely to match. Stem cells from the cord blood can be used for the new born, their siblings and other relatives. Patients with genetic disorders like cystic fibrosis need cells from sibling. There is 1 in 4(25%) chance that any of sibling will have inherited the same two sets of HLA genes as the patient. For a parent to be matched, with the patient, both parents must, by chance have some HLA genes in common with each other. The blood or the cheek swab (saliva) is tested for HLA TYPE for potential donor.

Chronic Graft versus host disease (GVHD) which includes dark skin rash, dry or thickened skin, loss of appetite etc. develops after 100 days of transplant usually, but rarely before 3 months after transplant. Up to 80% success results and more than 5 plus year survival rate has been identified owing it to the compatibility of immune system. The published data shows 23 years of cryopreservation of cord stem cells with more storage life for decades. The damage or failure of stem cells is attributed to DNA damage including telomere shortening, DNA replication and failure of repair.

In treating craniofacial deformities, craniofacial fractures or neoplastic lesions, traditional craniomaxillofacial reconstructive surgery techniques along with application of tissue enigineering and regenerative medicine provide long term adequate results avoiding the sequelae of tissue detrimentation. Studies have also shown that regeneration of bone in critical sized defects with periosteum preservation from a self assembling peptide nanofiber hydrogel with iPSCs. The results showed a marked increase in bone volume after 2–4 weeks with a nanofiber hydrogel scaffold with presence of medullary cavities and capillaries. This study suggests that auto-transplantation of osteoprogenitor cells derived from iPSCs combined with a suitable scaffold would be a good therapy for calvarial bone regeneration [11, 12].

Mesenchymal stem cells have the capacity of modulating the immune response and promote tissue regeneration. They can be harvested from many tissues such as skin, pancreas, heart, brain, lung, kidney, cartilage, tendon and teeth, with bone marrow and adipose tissue being the most common sources. The combination of recombinant human bone morphogenic protein-2&7 with mesenchymal stem cells in correction of cleft alveolus and their long term results is yet to be established [13, 14].

Bone marrow stem cells are considered as gold standard for bone regeneration. These stem cells have potential for osteogenic and chondrogenic differentiation. Bone marrow stem cells are obtained by bone marrow aspiration under local/general anesthesia at posterior superior iliac spine, sternum or by in-vitro cultivated bone marrow stem cells. It has been noted that the in-vitro cell population is less potent than the bone marrow aspirate. It is composed of both mesenchymal and hematopoetic stemcells. The disadvantages of bone marrow stem cells is that, there is an increased risk of surgical infection, donor site pain due to invasive technique, volume deficiency in larger defects which need combination of in-vitro culture cell population which is expensive. Conversely, adipose derived stem cells are more readily available as source and can be rapidly expanded [10].

Several in-vivo studies on bone defect regeneration after cyst enucleation, alveolar cleft sugeries, maxillary sinus floor elevation and augmentation using bone marrow stem cells showed favorable results with increased bone formation compared to traditional methods. It was observed that a scaffold free approach to reconstruction with bone marrow stem cell is safe for alveolar cleft repair, but not indicated in large cleft deficiencies. Bone marrow stem cell populations with in vivo activity along with demineralized bone matrix, platelet derived growth factor in tandem with tricalcium phosphate/hydroxyapatite or platelet rich fibrin composites generating bone repair mechanisms [11, 14].

Adipose derived stem cells are a promising alternative to bone marrow stem cells or traditional autogenous bone grafts. Comprising a high cell to volume than other stem cell categories, adipose stem cells are less sensitive to aging, easy to harvest and apply (isolated stromal vascular fraction) enriched with potent growth factors for improved results. Adipose derived stem cells can directly differentiate into osteoblasts and produce chemokines that are useful in facilitating the forming of endogenous stem cells to the site of bone defect. These stem cells can survive in hypoxic environment unlike mesenchymal stem cells by secreting vascular endothelial growth factor, platelet derived growth factor which promote blood vessel formation and enhance hematopoietic cells to allow the exchange of oygen, nutrients, wastes and growth factors necessary for cell survival. ADSC are negatively impacted by donor age in older population.

The stromal vascular fraction is a single source of a diverse population of cells that include multi-potent stem cells, progenitor cells, endothelial cells, stromal cells, pericytes, peri adipocytes, hematopoetic stem cells and macrophages. This holds a promising future direction in cleft palate and craniofacial bone reconstruction and regeneration [15]. In one clinical trial, stromal vascular fraction/adipose derived stem cells used in maxillary sinus floor elevation showed higher bone mass along with blood vessel formation compared to the control group with only adipose derived stem cells usage. ADSC in stromal vascular fraction have shown plasma membrane derived vesicles in the micro environment which establish inter cellular communication due to secretion of angiogenic molecules like FGF2, PDGF, VEGF, MMP-2, MMP-9 and osteogenic molecules like BMP2, RNA and micro RNA that impact the link with neighboring cells and the whole body. Overall, the ADSC have proved to be a multiple benefactor in safety, application and multiple cell transformation which can be collected in larger amounts in one step surgical procedure which decreases rate of infection.

ADSC are obtained through liposuction aspirate or resection of tissue fragments (buccal pad of fat). There are case reports with successful application of in vitro cultivated ADSC in alveolar cleft reconstruction, that wer seeded with demineralized bovine bone mineral and autologous bone. ADSC transferred by vehicular delivery through biphasic bone substitutes such as hydroxyl apatite-tricalcium phosphate scaffolds, poly-L-lactic acid scaffolds and bilaminate fibrin-agarose hydrogels showed significant bone regeneration compared to autologous bone grafting alone [16].

5.2 Tooth derived stem cells

Tooth derived stem cells are an interesting option in repairing bone defects of oral and dental tissues. These cells possess phenotypic characteristics similar to those of BMSCs, and they have the ability to self-renew and differentiate into multiple cell lineages, which are able to form the dentin-pulp structure when transplanted into immuno-compromised animal models. There are five classes of mesenchymal cell populations such as 1) dental pulpal stem cells 2) exfoliated deciduous teeth stem cells 3) periodontal ligament stem cells 4) dental follicle progenitor stem cells 5) stem cells from apical papilla. Of the above 5, stem cells from deciduous teeth (SHEDs) are easily extracted and isolated. They have high levels of immune stimulating and modulating chemokines, broad and multiple differentiation profile and strong proliferative capacity.

Human exfoliated deciduous teeth (SHEDs) are commonly isolated from patients between 5 and 12 years and are rich in post natal stem cells which could be induced into odontoblasts, osteoblasts, myocytes, adipocytes, and neuron-like cells. Dentin and bone could be formed when the cells are transplanted with bioactive materials in vivo. In addition, tooth derived stem cells participate in the repair and regeneration of non-dental tissues; in fact, these cells can differentiate into various types of cells, including neuron, hair follicle, hepatocyte, and cardiomyocyte like cells. Hydrogels may be a good option as a carrier for bone regeneration due to the osteoconductive characteristics of seeded MSCs as well as other advantages, such as injectability.

Dental pulpal stem cells are isolated from third molar extraction sites from teenage young adults. Both dental pulp stem cells and SHEDs are equally potent in cell regeneration and cultures with high concentration of secretomes (soluble paracrine signaling molecules) which allow for their immunomodulatory, angiogenic and neurogenic activities in vivo. SHEDs have been shown to form calvarial bone in critical size defect experiment as compared to other odontogenic tissue derived cell lines in an FGF-2 primed collagenous hydrogel deprived of oxygen, exhibiting markedly increased intramembranous ossification. Human derived dental pulp stem cells with collagen scaffold have the capacity of mature bone formation in calvarial defects with no graft rejection [17, 18, 19, 20].

In vivo implantation of the porous composite scaffolds within a critically sized calvarial defect in a rat showed near complete osseous closure of the defect over 6 weeks. An in vitro amplification for harvested MSCs is almost a necessity due to the relatively low numbers of harvested cells (1 MSC/10 4–10 6 stromal cells).

Bone marrow stromal stem cells have been studied in repair of auricular cartilage and craniofacial defects, when embedded in collagen scaffold [21]. Human adipose derived stem cells (h- ADSC) without any medium were able to correct skeletal defects which clearly showed a bone turn over within 2 weeks and a stimulation of the host’s reparative process. It was also noted that the bone morphogenic protein (BMP) modulated the h- ADSC through signaling during bone repair [22, 23].

Cranial suture stem cells (SuSC) isolated from calvarial sutures expressed Axin2, a marker to identify slow-cycling stem cells, which showed the ability of skeletal and cartilage repair. Direct engraftment of sutural stem cells (SuSC) to bone defect provided the benefits for cartilage repair through alteration of BMP signaling, leading the role of these cells in intramemebranous bone formation [24].

Several studies have been conducted in rabbit models for mandibular reconstruction with precise defects by integrating scaffolds such as polyether-ether-ketone (PEEK), fibrin glue, with ADSCs and MSCs transcripted with RUNX2 factor showed satisfactory promising results in terms of increase in new bone thickness, volume, compressive resistance, bone mineral density and content with good masticatory load strength [16, 23, 25].

Preliminary clinical studies have shown successful reconstruction with the combination of autologous bone grafts and human bone derived mesenchymal stem cells (BMSC) followed by distraction osteogenesis, dental implants and prosthodontic restoration. A clinical trial conducted by Gjerde et al. on 11 patients with posterior alveolar ridge resorption, evaluated mandibular regeneration using BMSCs without any additional factors like growth factors or stimulants or scaffolds. The result of this study showed successful ridge augmentation [26].

More clinical studies and trials are anticipated in mandibular and craniofacial reconstruction with larger defects using stem cells which could minimize the morbidity due to autologous bone grafting as well as provide long term results and enhance better living of patient.

6. Role of mesenchymal stem cells in craniofacial deformities/head and neck diseases

Majority of the craniomaxillofacial/head and neck anatomic region are formed from mesenchymal cells. Mesenchymal stem cells derived from dental and nondental sources have been effectively used for regeneration in maxillofacial region like regeneration of periodontium, salivary gland, repair of cleft lip and palate and craniofacial regeneration. These cells promote tissue regeneration and wound healing through synergistic downregulation of proinflammatory cytokines and increased production of soluble factors with antioxidant, anti apoptotic and proangiogenic properties. In oral wounds, they exhibit increased re-epithelialization, cellularity, intracellular matrix formation and neoangiogenesis, thereby accelerate wound healing. Hence mesenchymal stem cell therapy is a promising modality in healing soft tissue and hard tissue wounds of craniofacial region [7, 27].

Adipose cells with appropriate shaped scaffold can be used for reconstruct stem cells isolated from dental pulp has a potential to differentiate into osteoblasts and are a good source for bone formation. Stem cells from oral and maxillofacial region sub sites can be combined with bone marrow stem cells to correct larger defects. Oromaxillofacial bone tissue repair with stem cells was done using collagen sponge scaffold and dental pulp stem cells [9].

Scaffold free tissue constructs to close the critical size bone defects can be used in the form of microspheres. It was found that, osteogenically differentiated microspheres with outgrowing cells can be used to fill up bone defects. This new procedure has added advantage of permitting the transplantation of more cells and better integrity compared with cell suspensions or gels ion of soft tissues. Autologous fibrin glue that holds the cells in place was prepared by cryoprecipitation. This successful technique has given new rays of hope that ADSCs can be used for difficult reconstructive procedures of craniofacial defects [9].

Mesenchymal stem cells (MSCs) are multipotent stromal cells that are present in most adult connective tissues. MSCs have been widely used in stem cell transplantation, tissue engineering, gene therapy, and immunotherapy. These cells express CD105, CD73, andCD90, and are not able to express CD45, CD34, CD14, orCD11b, CD79α or CD19 antigens. In addition, they are able to differentiate into at least 3 cell lineages (immune modulatory, angiogenesis and antiapoptosis effects) in vitro, including chondroblasts, osteoblasts, and adipocytes.

MSC reduce IL-6, tumor necrosis factor-α (TNF-α), and IL-1β levels, 3 days after fracture. This process leads to a better regeneration by limiting tissue injury and inhibiting the progression of fibrosis. The production of inflammatory cytokines, including TNF –alfa, IL-6, IL-12p 70, and IFN-gamma, by macrophages is significantly suppressed by MSCs, while the production of anti-inflammatory cytokines like IL-10 and IL-12p40 is increased. Possibly PGE2 is the key mediator for this process [1]. The anti-apoptotic effect of MSCs could also accelerate the process of bone healing. It has been suggested that faster bone healing with MSC transplantation may be especially correlated with lower levels of TNF-α expression in the callus. This may favor bone formation since it has been reported that TNF-α can have pro-apoptotic effects on osteoblasts [1, 7].

6.1 Tissue engineering approaches with stem cells

In larger bone defects the local injection of stem cells is ineffective. Controlled delivery of MSCs to the desired site is achieved by three ways [2]. Delivery of cells within injectable or prefabricated scaffolds, [3] co-delivery of cells with osteoinductive growth factors or co-culture with other cell types, and [4] Delivery of cells within a 3D dynamic environment. A refabricated bone requires 3 elements, scaffolds or carriers (a 3D support), endothelial growth factors9stimulation of neovascularisation and provision of blood supply) and, MSCs and other growth promotion factors (stimulus for osteoinduction and recruitment of endogenous MSCs). An ideal scaffold/carrier should have four charecteristics, including osteogenesis, osteoincorporation, osteoinduction and osteoconduction [10].

Co-delivery of mesenchymal stem cells with prefabricated 3 dimensional scaffolds along with growth factors that possess properties of osteogenesis, osteoincorporation, osteoinduction, osteoconduction yields better results.

Future directions: A team of professionals including stem cell biologists, molecular biologists, geneticists, polymer and materials scientists, mechanical engineers and clinicians with knowledge of oral and maxillofacial disorders is needed to develop the field of craniofacial tissue engineering.

Though the stem cells and gene therapy have been used in experimental animal studies, it is a major challenge to accomplish regeneration of tissues and vascularity in larger craniofacial defects, as the cells must be within 100mu.m of an oxygen source to survive. In addition to vascular supply, accurate craniofacial reconstruction demands production of tissue interface to repair structures such as joint, tooth and muscle attachments. Use of gene transfer to engineer a cell in producing protein for tissue repair overcomes limitations of recombinant protein therapy in craniofacial regeneration. Somatic cells can be genetically corrected and re-programmed into iPS stem cells that in turn differentiate into disease free cells. Gene therapy in combination with iPS cell technology has great potential use in treating congenital disorders.

Application of stem cells in craniofacial regeneration and reconstruction should be transmuted from animal models to more number human case studies and clinical trials since substantial evidence is available through animal model studies regarding their results in craniofacial regeneration. Targeted tissue engineering therapy for reconstruction of defects and deformities in various sub-units of cranio-facial skeleton with the following illustrated methodology can yield more promising results in the future.- Calvarial bone regeneration through induced pluripotent stem cells delivered by hydrogel injectable system; Cartilagenous regeneration within nasal complex with cranial suture derived stromal stem cells; Maxillary and palatal bone regeneration by mesenchymal (adipose or bone marrow derived) stem cell delivery; Mandibular defect regeneration by polyether ether ketone (PEEK) scaffold delivery of mesenchymal stem cells. Particularly of more interest is the potency of developing induced pluripotent stem cells into specific cell lineage of requirement by selectively tuning the gene expression through genetic engineering. So far fundamentally, ADSCs and BMSCs have been successful as stem cell lineages in both pre-clinical and human clinical trials, more so are ADSCs in terms of bone regeneration. Stem cells have the dual capacity as cell based carrier for drug delivery as well as gene therapy. Insightful further research is required to understand the role of stem cells in cancer therapies, with the eventual goal of eliminating the residual disease and recurrence.

Summary

Autologous bone grafting has been the gold standard so far in the reconstruction of craniofacial defects and deformities. Future direction must point out towards application of stem cells for reconstruction of craniofacial defects and deformities of larger volume, thus minimizing the donor site morbidity caused by autologous hard and soft tissue grafting.