Current Advances in Mandibular Condyle Reconstruction

The temporomandibular joint, like any other synovial joint, can be the subject of severe degenerative pathological conditions as well as fracture and ankylosis. Advanced conditions may require rib or hip grafts, allografts, or total joint replacement. All current approaches suffer from inherent shortcomings and the search continues for a new approach to reconstruct the mandibular condyle with minimal or no side effects. Stem cell-based tissue engineering approach to reconstruct the mandibular condyle has long been introduced; however its potential clinical application requires long and costly dedicated research programs. Other therapeutic physical approaches to enhance tissue regenerative capacity have also been proposed, however their potential application needs further attention and investigation.


Introduction
The temporomandibular joint, like any other synovial joint, can be the subject of severe degenerative pathological conditions as well as fracture and ankylosis. Advanced conditions may require rib or hip grafts, allografts, or total joint replacement. All current approaches suffer from inherent shortcomings and the search continues for a new approach to reconstruct the mandibular condyle with minimal or no side effects. Stem cell-based tissue engineering approach to reconstruct the mandibular condyle has long been introduced; however its potential clinical application requires long and costly dedicated research programs. Other therapeutic physical approaches to enhance tissue regenerative capacity have also been proposed, however their potential application needs further attention and investigation.

Clinical indication
Articular joints have a poor innate ability to regenerate following either injury or disease. Among these diseases that affect articular joints is arthritis. In Canada, arthritis is the leading cause of work disability, with an economic cost of $4.4 billion in 1998 alone [1]. Statistics Canada reports estimated that 6 million Canadians will suffer from some form of arthritis by 2026, a significant increase from the current prevalence of four million Canadians [2]. The temporomandibular joint (TMJ) connects the mandible to the skull and is vital for speech, chewing, and swallowing.It is comprised of a mandibular condyle and an articular disk. TMJ is susceptible to arthritis, fractures, ankylosis, and dysfunctional syndromes that affect over 10 million individuals in North America [3][4][5][6][7][8][9]. To date, artificial joint replacement is considered the standard therapeutic procedure for degenerated TMJ, but this treatment approach has a high cost and non-predictive outcome [10]. According to the Canadian Joint Replacement Registry, a total of 97,671 patients had different joint replacements between years 2007-2010 [11].It has been reported that about 10% showed foreign body response to TMJ metal replacement with allergic reaction to metal [12]. Consequently, developing effective methods to replace articular condyle are of paramount importance to current/modern society. This book chapter discusses in detail contemporary methods and future directions of mandibular condylar reconstruction.

Mesenchymal stem cells
Mesenchymal stem cells (MSCs) are increasingly being used in joint tissue engineering research [13][14][15][16][17][18][19]. Tissue engineering ofmandibular condyle as a whole has been proposed in the literature; however an in-vivo utilization of this technique is in need of further investigation based upon compelling evidence from pilot data [15][16][17][18][19][20][21][22]. Some limitations to MSCs based therapy include the extended time needed in the laboratory to expand them and differentiate them into chondrogenic and osteogenic lineages. An improved approach to enhance the expansion and differentiation of MSCs is highly demanded. Also, understanding MSCs differentiation process and their characterization must be achieved before they can be used safely and effectively in articular joint replacement.
The current approach used to tissue engineer articular constructs involves conditioning with some type of mechanical stress. Existing mechanical conditioning techniques to enhance engineered tissues are in the form of bioreactors, BioFlex mechanical modulation technologies (Flexercell), and Instron machines. However, these approaches are short of clinical application should the engineered tissue require more mechanical modulation after in-vivo implantation for functional use.

Low intensity pulsed ultrasound
Low intensity pulsed ultrasound (LIPUS) therapy stimulates stem cell growth and differentiation [20,23-24]. We have shown in a pilot study in rabbits that LIPUS may enhance tissue engineered mandibular condyles. This compelling preliminary data needs to be validated in a statistically determined study design. Moreover, there is increasing supporting data in the literature that the stimulatory effect of LIPUS on cell expansion and differentiation is dose dependent. The LIPUS is considered the preferred method of mechanical stimulation, also known as "preferred bioreactor" [25].

Articular condyle
An articular condyle consists of articular cartilage and subchondral bone ( Fig. 1) [20]. Despite a common developmental origin from mesenchyme, the articular cartilage and subchondral bone have two distinct adult tissue phenotypes with few common morphological features. However, both tissues are structurally integrated and function in harmony to withstand mechanical loading up to several times the body's weight [26]. In osteochondral defects, bone regeneration can readily occur in the presence of an adequate blood supply up to a certain bony defect size. In contrast, articular cartilage has a poor capacity for self-regeneration. Furthermore, once articular cartilage is damaged, it undergoes degenerative events such as loss and/or destruction of key structural components, including type II collagen and proteoglycans. The poor capacity of cartilage for self-regeneration is likely attributed to the paucity of tissue-forming cells (i.e., chondrocytes) [27] and the lack of access to systemically available mesenchymal stem cells because the cartilage tissue is avascular. Thus, the self-regenerating capacity of articular cartilage is limited due to the sparsely available chondroprogenitor cells and/or the scant local mesenchymal stem cells that are habitual residents. Importantly, the articular cartilage is devoid of a nerve supply. Thus, articular cartilage injuries are often not accompanied by joint pain until the damage has progressed to involve the subchondral bone, which contains rich nerve supply [28]. In many of these disorders, structural damage of the TMJ necessitates surgical replacement.

TMJ replacement
The current TMJ replacement techniques utilize bone/cartilage grafts, muscles and artificial materials [9,[29][30]. Despite certain level of reported clinical success, autografts are associated with donor site morbidity such as discomfort in ambulation, sensorial loss over the donor region, scars, and contour deformity when bone is harvested from the iliac bone. Also, predictability of clinical outcome of autografts is reported to be substandard with graft overgrowth in 10% of patients and undergrowth in 57% of patients, and a relatively high incidence of re-operation with 23% of patients requiring re-grafting [31][32][33][34].Alternatively, alloplastic and xenoplastic grafts are associated with potential transmission of pathogens and immunorejection [35][36][37].The failure rate of using alloplastic grafts to reconstruct the TMJ has been reported to reach 30% [38]. To date, there is no consistent clinically-effective and safe method to replace the TMJ or mandibular condyle.

Biological replacement of mandibular condyle
Biological replacement efforts for reconstruction of the mandibular/articular condyles have included using osteoblasts and chondroblasts/chondrogenic cells from different tissue/cell sources [15][16][17][18][19][20][21][22][38][39][40][41]. However, these efforts have been limited by several obstacles including: a) scarcity of stem cells with the capacity to differentiate into chondrogenic and osteogenic cells, b) different bone ingrowth patterns [37], c) different rates of the scaffold degradation compared to matrix production [15], and d) inferior mechanical properties of the regenerative tissue for clinical use [40]. Moreover, the integration of tissue engineered constructs for osteochondral repair requires an inordinate amount of time (3-6 months in rabbit femur heads [21],6-12 months in horses [41], and up to 9 months in sheep [19]). Regeneration of articular joints utilizing a cell-free scaffold by cell homing to the area shows some success [18]. However, this process did not provide full articular condyle replacement. In addition, this proof of principle lasted 9 weeks to obtain some articular joint regeneration in rabbits, which translates to 9 to 12 months in humans, given the difference in metabolism between the two species [42]. This lengthy time of manipulation can be complicated by tissue culture problems such as infection. Another attempt to tissue engineer mandibular condyle using porcine stem cells demonstrated bone formation in-vitro; however there was no attempt or success in translating this technique into in-vivo utilization [43]. A similar recent study demonstrated the possibility of tissue engineering a complete mandibular condyle in-vitro; however in-vivo utilization of this technique has yet to be studied [44]. Interestingly, this study highlighted the importance of bioreactor in stem cell expansion and differentiation [44]. It was first reported that tissue engineered osteochondral constructs from MSCs can be shaped into human-size mandibular condyles while maintaining the shape and size after extended period of in-vivo implantation [15,17,18]. Not only these constructs demonstrate MSCs-driven formation of osteochondral tissue-like histologically, but also both tissue types showed good histological integration attributed to the use of the same scaffolding material in both layers, and thus avoiding the potential fibrous tissue infiltration between the two layers usually observed in composite constructs [15,17,18].Our team was the first to report on the possibility of engineering condyles from stem cells [15,17,18] (Figure 2). Although most of the recent studies, including ours, are focused on engineering scaffolds in the shape of mandibular or articular condyles [15,17,18,44], future research is needed to implement tissue engineered condyles into clinical application and to demonstrate functional integration. It is well known that inadequate mechanical strength is considered a major impediment to cartilage tissue engineering [45,46]. The material properties of tissueengineered cartilage constructs are in the range of kilopascals [47], which are orders of magnitude lower than normal articular cartilage (in the range of megapascals) [48][49][50][51][52][53]. Different techniques have attempted to improve the quality of tissue-engineered articular joints. Pulsed electromagnetic fields (PEMF) have been shown to increase chondrocyte and osteoblast-like cell proliferation [54,55]. Bioreactors including LIPUS enhance the material properties of tissue-engineered cartilage constructs [25, 56,57]. Cyclic compressive loading induces phenotypic changes in cartilaginous and osseous tissues in cell culture, scaffolds, and in-vivo [58][59][60][61][62][63][64][65][66][67][68][69][70]. Also, mechanical stimulation enhances the expression of vascular endothelial growth factor (VEGF) which is important for angiogenesis and bone formation in the mandibular condyles [71]. These important discoveries support the potential for clinical application of different forms of mechanical stimulation to enhance tissue-engineered joint tissues.

Low intensity pulsed ultrasound (LIPUS)
Low intensity pulsed ultrasound (LIPUS) is a form of mechanical stimulation that has been used to enhance healing of fractured bone and other tissues. Details about the current literature and the potential use of LIPUS for better autologous stem cell based mandibular condyle (ASCMC) will be discussed below. It is clear that there is a vital need for an approach to enhance stem cell expansion and differentiation for tissue engineering of articular condyles. LIPUS can be an effective tool to enhance tissue-engineering of mandibular condyles for many reasons. Importantly, LIPUS is the preferred method of mechanical stimulation, also reported as "preferred bioreactor" [25] as it enhances angiogenesis [20, [72][73][74][75][76].This is especially relevant because vasculature is required to integrate the engineered tissue with the native surrounding tissues [77]. Recent studies showed that LIPUS enhances stem cell expansion and differentiation in tissue culture [78,79]. Also, LIPUS has been shown to enhance periosteal cell expansion [79] and stimulate bone marrow stem cells (BMSCs) expansion and differentiation into chondrogenic lineage [78,[80][81][82][83].The matrix production and proliferation of the intervertebral disc cells in culture has been shown to be enhance by LIPUS [82]. In addition, LIPUS enhances osteoblast matrix formation [796,83] and minimizes apoptosis of human stem cells in-vitro [84]. The optimum LIPUS application time in bone fracture healing has been identified [85]; however, the optimum LIPUS treatment timing in articular condyle replacement is yet to be studied.Despite recent studies that have shown that the stimulatory effect of LIPUS in tissue culture is dose-dependent (treatment time) [23,24,75,78,[86][87][88], the use of LIPUS has not resulted in any severe adverse events in tissue culture [88], human or animal models [89][90][91][92]. Our research has demonstrated that LIPUS can enhance stem cell expansion in monolayers [20-23-24] (Figure 3).There was an increase in cell number after LIPUS application for 20 minutes per day for 3 weeks. A future projectcan aim to optimize using LIPUS to enhance cell proliferation to a significant level that may justify its routine use in tissue engineering. In addition, LIPUS enhances expression of bone morphogenetic proteins from pluripotent cells [88]. Moreover, we have shown that LIPUS application for 20 minutes per day for 4 weeks increased the expression of collagen II and osteopontin expression in osteogenic-induced differentiation of stem cells (P<0.05)[ Figure 4 and Table A   Also, LIPUS application to gingival stem cells statistically increased the gene expression of alkaline phosphatase (ALP) in tissue culture ( Figure 5) [88].  Finally, we have shown that LIPUS enhances tissue-engineered mandibular condyles in a pilot study invivo [20] (Figures 7-13). This was confirmed qualitatively by MicroCT scanning, histological evaluations (safranin O and Von Kossa staining) (Figures 9-12) as well as quantitatively by histomorphometric analysis (Figure 13).

Mechanical stress and intracellular signaling
There is growing evidence in the literature that integrins are promising candidates for sensing extracellular matrix-derived mechanical stimuli and converting them into biochemical signals [93][94][95][96]. Integrin-associated signaling pathways include an increase in tyrosine phosphorylation of several signaling proteins, activation of serine/threonine kinases, and alterations in cellular phospholipid and calcium levels [97][98]. These events are associated with the formation of focal adhesions, which contain structural proteins such as Src, and Shc. Focal adhesions act as a bridge to link integrin cytoplasmic domain to the cytoskeleton and activate integrinassociated signaling pathways, such as the mitogen-activated protein kinase (MAPK) pathway [99] and the Rho pathway [100][101]. Rho and its downstream target Rho kinase/Rho-associated coiled-coil-containing protein kinase (ROCK) [102] are involved in the reorganization of cytoskeletal components [99], [102][103]. It has been recently reported that β1 integrin plays predominant roles for shear-induced signaling and gene expression in osteoblast-like MG63 cells on FN, COL1, and Laminin (LM) and that αvβ3 also plays significant roles for such responses in cells on fibronectin (FN). The β1 integrin-Shc association leads to the activation of ERK, which is critical for shear induction of bone formation-related genes in osteoblast-like cells [103]. Moreover, α5β1 integrin is expressed by chondrocytes [104] and it plays an important role in mechanically enhanced cartilage tissue engineering. Furthermore, integrins were found to be responsible for ultrasound-induced cell proliferation. It has been suggested that integrins act as mechanotransducers to transmit acoustic pulsed energy into intracellular biochemical signals inducing cell proliferation [105]. It has been reported recently that LIPUS activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes [106] as well as increases FAK, ERK-1/2, and IRS-1 expression of intact rat bone cells [107]. This has yet to be investigated in MSC derived chondrocytes and in osteoblasts-like cells.

Conclusion
The literature supports that mechanical stress, for example LIPUS have a stimulatory effect on stem cell expansion and differentiation as well as enhancing stem cell matrix production in-vitro and in a pilot study in-vivo in rabbits. However, these results need to be validated in a large scale in-vivo.We are now poised to prove these effects in a large scale study. Although the optimum mechanical stimulation, for example LIPUS treatment time, for bone fracture healing is well documented, the corollary for enhancing autologous stem cell based replacement of mandibular condyles has not been investigated. This represents a major gap of knowledge in the field of tissue engineering considering the numerous positive utilizations of mechanical stimulation as well as LIPUS reported in the literature. Overall, the current literature and knowledge developed through our and others' research has the potential to increase our understanding of the details of LIPUS induced chondrogenesis and osteogenesis and how to utilize LIPUS to enhance articular joint replacement using MSCs. Furthermore, this knowledge could give rise to a novel cell-based therapy for replacement of mandibular condyles as well as other tissue types.