Morphologic and histologic characterization of sheep and porcine TMJ as large animal models for tissue engineering applications.

OBJECTIVE: The aim of this study was to compare and characterize the structural and ultrastructural organization of the temporomandibular joint (TMJ) between two large animal models for use in the development of tissue engineering strategies. MATERIALS AND METHODS: Whole TMJs from sheep and pigs were evaluated with micro-computed tomography (µCT) for morphology and quantitative analyses of bone parameters. Histological examination was performed on the TMJ disc and its attachments to investigate regional distribution of collagen, elastin, and glycosaminoglycans (GAGs). RESULTS: µCT analyses demonstrate higher bone mineral density (BMD) in the temporal fossa compared to the mandibular condyle in both species, with this variable being significantly higher in sheep than pig. Quantitative morphometry of the trabecular condyle reveals no statistical differences between the species. Histology demonstrates similar structural organization of collagen and elastin between species. Elastin staining was nearly twofold greater in sheep than in the pig disc. Finally, Safranin-O staining for GAGs in the TMJ disc was localized to the intermediate zone in the sheep but was absent from the porcine disc. CONCLUSIONS: Our findings show some important differences in the pig and sheep TMJ µCT variables and histology and composition of the disc and discal attachment. These disparities likely reflect differences in masticatory and TMJ functional loading patterns between the two species and provide insights into large animal models towards human applications. CLINICAL RELEVANCE: As with the established pig model, the sheep is a suitable large animal model for TMJ research such as regenerative strategies, with specific considerations for design parameters appropriate for human-analog applications.

Comparison of the Trueness of Fits of the Biphasic Transverse Isotropic and Kelvin Models to the Tensile Behavior of Temporomandibular Joint Disc.

This technical brief explores the validity and trueness of fit for using the transverse isotropic biphasic and Kelvin models (first and second order generalized) for characterization of the viscoelastic tensile properties of the temporomandibular joint (TMJ) discs from pigs and goats at a strain rate of 10 mm/min. We performed incremental stress-relaxation tests from 0 to 12% strain, in 4% strain steps on pig TMJ disc samples. In addition, to compare the outcomes of these models between species, we also performed a single-step stress-relaxation test of 10% strain. The transverse isotropic biphasic model yielded reliable fits in reference to the least root mean squared error method only at low strain, while the Kelvin models yielded good fits at both low and high strain, with the second order generalized Kelvin model yielding the best fit. When comparing pig to goat TMJ disc in 10% strain stress-relaxation test, unlike the other two Kelvin models, the transverse isotropic model did not fit well for this larger step. In conclusion, the second order Kelvin model showed the best fits to the experimental data of both species. The transverse isotropic biphasic model did not fit well with the experimental data, although better at low strain, suggesting that the assumption of water flow only applies while uncrimping the collagen fibers. Thus, it is likely that the permeability from the biphasic model is not truly representative, and other biphasic models, such as the poroviscoelastic model, would likely yield more meaningful outputs and should be explored in future works.

Trueness of Fit of Biphasic Transversely Isotropic Parameters Model in the Porcine Temporomandibular Joint Disc and Mandibular Condylar Cartilage and Regional Dependence.

Temporomandibular joint (TMJ) disorders (TMDs) are not well understood and the mechanical differences between the regions of the mandibular condylar cartilage (MCC) and the TMJ disc have not been thoroughly compared. As of now, there are no commercially available regenerative therapies for the TMJ. Elucidating the mechanical properties of these two structures of the articulating joint will help future efforts in developing tissue engineering treatments of the TMJ. In this study, we evaluate the compressive properties of the porcine disc and mandibular condylar cartilage by performing unconfined compression at 10% strain with 4.5%/min strain rate. Punches (4 mm biopsy) from both tissues were taken from five different regions of both the MCC and TMJ: anterior, posterior, lateral, medial, and central. Previously, theoretical models of compression in the porcine tissue did not fit the whole ramp-relaxation behavior. Thus, the data stress-relaxation was fitted to the biphasic transversely isotropic model, for both the TMJ disc and cartilage. From the results found in the disc, it was found that the posterior region had the highest values in multiple viscoelastic parameters when compared to the other regions. The mandibular condylar cartilage was only found to be significantly different in the transverse modulus between the posterior and lateral regions. Both the TMJ disc and MCC had similar magnitudes of values (for the modulus and other corresponding compressive properties) and behavior under this testing modality.

Temporomandibular Joint Bioengineering Conference: Working Together Toward Improving Clinical Outcomes.

The sixth temporomandibular joint (TMJ) Bioengineering Conference (TMJBC) was held on June 14-15 2018, in Redondo Beach, California, 12 years after the first TMJBC. Speakers gave 30 presentations and came from the United States, Europe, Asia, and Australia. The goal of the conference has remained to foster a continuing forum for bioengineers, scientists, and surgeons and veterinarians to advance technology related to TMJ disorders. These collective multidisciplinary interactions over the past decade have made large strides in moving the field of TMJ research forward. Over the past 12 years, in vivo approaches for tissue engineering have emerged, along with a wide variety of degeneration models, as well as with models occurring in nature. Furthermore, biomechanical tools have become more sensitive and new biologic interventions for disease are being developed. Clinical directives have evolved for specific diagnoses, along with patient-specific biological and immunological responses to TMJ replacement devices alloplastic and/or bioengineered devices. The sixth TMJBC heralded many opportunities for funding agencies to advance the field: (1) initiatives on TMJ that go beyond pain research, (2) more training grants focused on graduate students and fellows, (3) partnership funding with government agencies to translate TMJ solutions, and (4) the recruitment of a critical mass of TMJ experts to participate on grant review panels. The TMJ research community continues to grow and has become a pillar of dental and craniofacial research, and together we share the unified vision to ultimately improve diagnoses and treatment outcomes in patients affected by TMJ disorders.

Are mTOR and Endoplasmic Reticulum Stress Pathway Genes Associated with Oral and Bone Diseases?

The purpose of this cohort study was to identify associations between combined oral and bone disease phenotypes and genes present in cell regulatory pathways. The studied pathways play important roles in cellular growth, proliferation, differentiation, and homeostasis. DNA samples extracted from whole saliva of 3,912 individuals were genotyped and these data analyzed according to dental caries experience, periapical lesions, periodontitis, osteoporosis, or temporomandibular joint discomfort. Samples were obtained from the Dental Registry and DNA Repository project at the University of Pittsburgh. Twenty-seven polymorphisms in eight genes related to mTOR or endoplasmic reticulum stress pathways were selected for genotyping. Allele frequencies and Hardy-Weinberg equilibrium were calculated. Analyses were performed comparing genotypes between affected and unaffected individuals for each phenotype, as well as for the associated phenotypes combined. For all analyses, we used the software PLINK with an alpha of 0.002. Borderline associations with multiple variants of several genes were found, suggesting that both pathways may be involved in the susceptibility to multiple conditions affecting the oral cavity and bones. When combining patients that had concomitant dental caries, periodontitis, and periapical pathology, several markers in RHEB showed statistically significant association. Multiple conditions affecting bone and teeth (i.e., dental caries, periodontitis, periapical lesion formation, and osteoporosis) appear to share similar underlying genetic etiological factors, which allow us to hypothesize that instead of individually, they should be studied in conjunction in human populations.

Potential trade-offs between biomineralization and immunity revealed by shell properties and gene expression profiles of two closely related Crassostrea species.

Species of the Ostreidae family are key ecosystem engineers and many of them - including Crassostrea gigas and Crassostreavirginica - are commercially important aquaculture species. Despite similarities in their morphology and ecology, these two species differ in their ability to defend against pathogens, potentially reflecting species-specific differential specialization of hemocytes on immune defense versus biomineralization. To test this hypothesis, we investigated the expression levels of immune- and biomineralization-related genes as well as mineralogical and mechanical properties of the shells and the calcium sequestration ability of the hemocytes of C. gigas and C. virginica The expression of biomineralization-related genes was higher in C. virginica than in C. gigas in multiple tissues including the mantle edge and hemocytes, while the expression of immune genes was higher in the hemocytes of C. gigas Hemocytes of C. virginica contained more calcium (stored intracellularly as calcium carbonate mineral) compared with those of C. gigas Analysis of the adult shells showed that the crystallinity of calcite was higher and the laths of the foliated layer of the shell were thicker in C. virginica than in C. gigas Mechanically, the shells of C. virginica were stiffer, harder and stronger than those of C. gigas Taken together, our results show that the species-specific differences in physiology (such as disease resistance and exoskeleton properties) are reflected at the cellular and molecular levels in the differential specialization of hemocytes on potentially competing functions (immunity and biomineralization) as well as different expression profiles of other tissues involved in biomineralization (such as the mantle edge).

Properties of the Temporomandibular Joint in Growing Pigs.

A subset of temporomandibular joint (TMJ) disorders is attributed to joint degeneration. The pig has been considered the preferred in vivo model for the evaluation of potential therapies for TMJ disorders, and practical considerations such as cost and husbandry issues have favored the use of young, skeletally immature animals. However, the effect of growth on the biochemical and biomechanical properties of the TMJ disk and articulating cartilage has not been examined. The present study investigates the effect of age on the biochemical and biomechanical properties of healthy porcine TMJs at 3, 6, and 9 months of age. DNA, hydroxyproline, and glycosaminoglycan (GAG) content were determined and the disks and condyles were tested in uniaxial unconfined stress relaxation compression from 10% to 30% strain. TMJ disks were further assessed with a tensile test to failure technique, which included the ability to test multiple samples from the same region of an individual disk to minimize the intraspecimen variation. No differences in biochemical properties for the disk or compressive properties at 30% stress relaxation in the disk and condylar cartilage were found. In tension, no differences were observed for peak stress and tensile modulus. The collagen content of the condyle was higher at 9 months than 3 months (p < 0.05), and the GAG content was higher at 9 months than 6 months (p < 0.05). There was a trend of increased compressive instantaneous modulus with age. As such, age-matched controls for growing pigs are probably appropriate for most parameters measured.

Regenerative Potential of Various Soft Polymeric Scaffolds in the Temporomandibular Joint Condyle.

PURPOSE: Biodegradable polymeric scaffolds have been used for tissue engineering approaches and can be used to regenerate temporomandibular joint (TMJ) tissues. Synthetic acellular polymeric poly(glycerol sebacate) (PGS) scaffolds and natural scaffolds made from gelatin are polymeric scaffold sponges that could provide a substrate for cell infiltration and remodeling. The authors studied the regenerative potential of these 2 scaffolds in addition to a bioactive signal, magnesium (Mg), in a novel fibrocartilage defect model in the goat mandibular condylar cartilage (MCC). Furthermore, in a departure from the pig model, the authors have started to develop the goat as a repeatable surgical model with easy access into the joint space in skeletally mature animals. MATERIALS AND METHODS: Bilateral osteochondral defects were created in the mandibular condyle of mature female Spanish Boer goats. A 1-mm diameter drill was used to create a trough defect on the articular surface. Four groups were evaluated: 1) an empty control without an implant, 2) PGS with Mg ions, 3) gelatin with Mg ions, and 4) gelatin with Mg ions and trimagnesium phosphate (TMP) powder. Goats were allowed to heal for 3 months, and then the tissues were harvested. RESULTS: The empty control group showed a thin fibrous layer growing within the defect. The PGS and gelatin sponge groups showed a cartilage layer with glycosaminoglycan and collagen type II and robust regeneration of the fibrous layer as exhibited by cell infiltration and collagen in the defect. TMP in the gelatin did not degrade and seemed to hamper healing. CONCLUSION: These results suggest that synthetic and natural sponges can provide a template for new tissue growth in the MCC of the TMJ. Furthermore, this study is the first to attempt to develop the goat as an in vivo TMJ tissue regeneration model.

Potential Indications for Tissue Engineering in Temporomandibular Joint Surgery.

PURPOSE: Musculoskeletal tissue engineering has advanced to the stage where it has the capability to engineer temporomandibular joint (TMJ) anatomic components. Unfortunately, there is a paucity of literature identifying specific indications for the use of TMJ tissue engineering solutions. The objective of this study was to establish an initial set of indications and contraindications for the use of engineered tissues for replacement of TMJ anatomic components. FINDINGS: There was consensus among the authors that the management of patients requiring TMJ reconstruction as the result of 1) irreparable condylar trauma, 2) developmental or acquired TMJ pathology in skeletally immature patients, 3) hyperplasia, and 4) documented metal hypersensitivities could be indications for bioengineered condyle and ramus TMJ components. There was consensus that Wilkes stage III internal derangement might be an indication for use of a bioengineered TMJ disc or possibly even a disc-like bioengineered "fossa liner." However, there was some controversy as to whether TMJ arthritic disease (e.g., osteoarthritis) and reconstruction after failed alloplastic devices should be indications. Further research is required to determine whether tissue-engineered TMJ components could be a viable option for such cases. Contraindications for the use of bioengineered TMJ components could include patients with TMJ disorders and multiple failed surgeries, parafunctional oral habits, persistent TMJ infection, TMJ rheumatoid arthritis, and ankylosis unless the underlying pathology can be resolved. CONCLUSIONS: Biomedical engineers must appreciate the specific indications that might warrant TMJ bioengineered structures, so that they avoid developing technologies in search of problems that might not exist for patients and clinicians. Instead, they should focus on identifying and understanding the problems that need resolution and then tailor technologies to address those specific situations. The aforementioned indications and contraindications are designed to serve as a guide to the next generation of tissue engineers in their strategic development of technologies to address specific clinical issues.

Decreased Temporomandibular Joint Range of Motion in a Model of Early Osteoarthritis in the Rabbit.

PURPOSE: Analysis of mandibular biomechanics could help with understanding the mechanisms of temporomandibular joint (TMJ) disorders (TMJDs), such as osteoarthritis (TMJ-OA), by investigating the effects of injury or disease on TMJ movement. The objective of the present study was to determine the functional kinematic implications of mild TMJ-OA degeneration caused by altered occlusion from unilateral splints in the rabbit. MATERIALS AND METHODS: Altered occlusion of the TMJ was mechanically induced in rabbits by way of a unilateral molar dental splint (n = 3). TMJ motion was assessed using 3-dimensional (3D) skeletal kinematics twice, once before and once after 6 weeks of splint placement with the splints removed, after allowing 3 days of recovery. The relative motion of the condyle to the fossa and the distance between the incisors were tracked. RESULTS: An overall decrease in the range of joint movement was observed at the incisors and in the joint space between the condyle and fossa. The incisor movement decreased from 7.0 ± 0.5 mm to 6.2 ± 0.5 mm right to left, from 5.5 ± 2.2 mm to 4.6 ± 0.8 mm anterior to posterior, and from 13.3 ± 1.8 mm to 11.6 ± 1.4 mm superior to inferior (P < .05). The total magnitude of the maximum distance between the points on the condyle and fossa decreased from 3.6 ± 0.8 mm to 3.1 ± 0.6 mm for the working condyle and 2.8 ± 0.4 mm to 2.5 ± 0.4 mm for the balancing condyle (P < .05). The largest decreases were seen in the anteroposterior direction for both condyles. CONCLUSION: Determining the changes in condylar movement might lead to a better understanding of the early predictors in the development of TMJ-OA and determining when the symptoms become a chronic, irreversible problem.

BMP-2-regenerated calvarial bone: a biomechanical appraisal in a large animal model.

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is gaining popularity in craniofacial applications. Calvarial defects are, under normal circumstances, subjected to only minimal levels of the biomechanical stresses known to play an important role in osteogenesis, yet regenerated calvarial bone must be capable of withstanding traumatic forces such that the underlying neurocapsule is protected. The aim of this study is to, for the first time, assess the biomechanical properties of calvarial bone regenerated with derivations of a commercially available rhBMP-2-based system. Standardized calvarial defects were created in 23 adult male canines. These defects were treated with rhBMP-2 on one of several carriers. After 24 weeks, the biomechanical properties of the rhBMP-2-generated bone were compared to those of controls with a modified punch-out test (Bluehill 2; Instron, Norwood, Mass) and compared using a paired nonparametric analyses (SPSS, 17.0, Chicago, Ill). In a previously published report, defects across all the rhBMP-2 therapy groups were observed to have a mean rate of 99.5% radio-opacity at 24 weeks indicating nearly full bony coverage of the calvarial defect (compared to 32.7% in surgical controls). For ultimate load, ultimate energy, and first peak energy, there were significant differences (P<0.05) with the control native bone having more robust biomechanical properties than the rhBMP-2-generated bone. We conclude from these findings that rhBMP-2-generated calvarial bone is significantly less protective against trauma than native bone at 6 months. Further investigation is required to assess the efficacy of rhBMP-2 in healing calvarial defects in the longer term.

Na+/H+ exchanger regulatory factor 1 (NHERF1) directly regulates osteogenesis.

Bone formation requires synthesis, secretion, and mineralization of matrix. Deficiencies in these processes produce bone defects. The absence of the PDZ domain protein Na(+)/H(+) exchange regulatory factor 1 (NHERF1) in mice, or its mutation in humans, causes osteomalacia believed to reflect renal phosphate wasting. We show that NHERF1 is expressed by mineralizing osteoblasts and organizes Na(+)/H(+) exchangers (NHEs) and the PTH receptor. NHERF1-null mice display reduced bone formation and wide mineralizing fronts despite elimination of phosphate wasting by dietary supplementation. Bone mass was normal, reflecting coordinated reduction of bone resorption and formation. NHERF1-null bone had decreased strength, consistent with compromised matrix quality. Mesenchymal stem cells from NHERF1-null mice showed limited osteoblast differentiation but enhanced adipocyte differentiation. PTH signaling and Na(+)/H(+) exchange were dysregulated in these cells. Osteoclast differentiation from monocytes was unaffected. Thus, NHERF1 is required for normal osteoblast differentiation and matrix synthesis. In its absence, compensatory mechanisms maintain bone mass, but bone strength is reduced.

Inductive, scaffold-based, regenerative medicine approach to reconstruction of the temporomandibular joint disk.

PURPOSE: A device composed of extracellular matrix (ECM) was investigated as an inductive template in vivo for reconstruction of the temporomandibular joint (TMJ) disk after discectomy. MATERIALS AND METHODS: A scaffold material composed of porcine-derived ECM was configured to mimic the shape and size of the TMJ. This device was implanted in a canine model of bilateral TMJ discectomy. After discectomy, 1 side was repaired with an ECM scaffold material and the contralateral side was left empty as a control. At 6 months after implantation, the joint space was opened, the joints were evaluated for signs of gross pathologic degenerative changes, and newly formed tissue was excised for histologic, biochemical, and biomechanical analysis. RESULTS: The results showed that implantation of an initially acellular material supported the formation of site-appropriate, functional host tissue that resembled that of the native TMJ disk. Furthermore, this prevented gross degenerative changes in the temporal fossa and mandibular condyle. No tissue formation and mild to severe gross pathologic changes were observed in the contralateral controls. CONCLUSIONS: These results suggest that an ECM-based bioscaffold could represent an off-the-shelf solution for TMJ disk replacement.

Potential of healing a transected anterior cruciate ligament with genetically modified extracellular matrix bioscaffolds in a goat model.

PURPOSE: Biological augmentation to heal a torn anterior cruciate ligament (ACL) has gained significant interest. This study examined the potential advantages of using extracellular matrix (ECM) bioscaffolds from galactosyl-α(1,3)galactose deficient pigs to heal the transected ACL. METHODS: In 16 skeletally mature goats, the ACL in the right hindlimb was transected and repaired. In 9 of these animals, an ECM sheet was wrapped around the injury site and with an ECM hydrogel injected into the transected site. The remaining 7 animals were treated with suture repair only. The left hindlimb served as a sham-operated control. RESULTS: After 12 weeks, the healing ACL in the ECM-treated group showed an abundance of continuous neo-tissue formation, while only limited tissue growth was found after suture repair only. The cross-sectional area of the ACL from the ECM-treated group was similar to sham-operated controls (n.s.) and was 4.5 times those of the suture repair group (P < 0.05). The stiffness of the femur-ACL-tibia complexes from the ECM-treated group was 2.4 times those of the suture repair group (P < 0.05). Furthermore, these values reached 48% of the sham-operated controls (53 ± 19 N/mm and 112 ± 21 N/mm, respectively, P < 0.05). CONCLUSIONS: The application of an ECM bioscaffold and hydrogel was found to accelerate the healing of a transected ACL following suture repair in the goat model with limited tissue hypertrophy and improvement in some of its biomechanical properties. Although more work is necessary to fully restore the function of the normal ACL, these early results offer a potential new approach to aid ACL healing.

Decellularized small intestine submucosa device for temporomandibular joint meniscus repair: Acute timepoint safety study.

Temporomandibular joint (TMJ) Meniscus removal is an option for the patient to regain full range of motion if the disc is irreversibly damaged or unable to be reduced. However, this procedure leaves the joint vulnerable to condylar remodeling and degeneration. We have shown that extracellular matrix (ECM) scaffolds remodel into a tissue with near native TMJ meniscus in previous studies. The next step towards clinical translation is to manufacture the ECM scaffold as a device under good manufacturing practices (GMP) and test it in a pre-clinical animal study under good laboratory practices (GLP). The primary objective of this study was to evaluate the in-vivo histopathological response to a Prototype GMP manufactured device made of decellularized porcine small intestinal submucosa (SIS), by observing for signs of surrounding tissue reaction to the device that are indicative of an adverse host response in comparison to an empty control at 21 days post-surgical implantation in a canine TMJ meniscus removal and implant model in a GLP setting. The conclusive findings were that the ECM device is safe for placement in the TMJ. After 21 days post implantation, histology of tissue surrounding the device and draining lymph nodes showed that the Prototype GMP device had no negative effects compared to the empty site (as evaluated by the board-certified veterinary pathologist). Furthermore, there was a lack of negative findings for clinical pathology (hematology and clinical chemistry), mortality, and body weight/weight change. Future studies will go to one year after implantation to show that the remodel device remains as a viable tissue with near native mechanical properties.

Inductive Remodeling of Extracellular Matrix Scaffolds in the Temporomandibular Joint of Pigs.

The temporomandibular joint (TMJ) disc is a fibrocartilaginous tissue located between the condyle of the mandible and glenoid fossa and articular eminence of the temporal bone. Damage or derangement of the TMJ disc can require surgical removal (discectomy) to restore function. Removal of the TMJ disc, however, leaves the joint space vulnerable to condylar remodeling and degradation, potentially leading to long-term complications. No consistently effective clinical option exists for repair or replacement of the disc following discectomy. This study investigates the use of an acellular scaffold composed of extracellular matrix (ECM) derived from small intestinal submucosa (SIS) as a regenerative template for the TMJ disc in a porcine model. Acellular SIS ECM scaffolds were implanted following discectomy and allowed to remodel for 2, 4, 12, and 24 weeks postimplantation. Remodeling of the implanted device was assessed by longitudinal magnetic resonance imaging (MRI) over the course of 6 months, as well as gross morphologic, histologic, biochemical, and biomechanical analysis (tension and compression) of explanted tissues (disc and condyle) at the time of sacrifice. When the scaffold remained in the joint space, longitudinal MRI demonstrated that the scaffolds promoted new tissue formation within the joint space throughout the study period. The scaffolds were rapidly populated with host-derived cells and remodeled with formation of new, dense, aligned fibrocartilage resembling native tissue as early as 1 month postimplantation. De-novo formation of peripheral muscular and tendinous attachments resembling those in native tissue was also observed. The remodeled scaffolds approached native disc biochemical composition and compressive modulus, and possessed 50% of the tensile modulus within 3 months postimplantation. No degradation of the condylar surface was observed. These results suggest that this acellular bioscaffold fills a medical need for which there is currently no effective treatment and may represent a clinically relevant "off-the-shelf" implant for reconstruction of the TMJ disc.

Hydrogel to guide chondrogenesis versus osteogenesis of mesenchymal stem cells for fabrication of cartilaginous tissues.

The ideal combination of hydrogel components for regeneration of cartilage and cartilaginous interfaces is a significant challenge because control over differentiation into multiple lineages is necessary. Stabilization of the phenotype of stem cell derived chondrocytes is needed to avoid undesired progression to terminal hypertrophy and tissue mineralization. A novel ternary blend hydrogel composed of methacrylated poly(ethylene glycol) (PEG), gelatin, and heparin (PGH) was designed to guide chondrogenesis by bone marrow derived mesenchymal stem cells (BMSCs) and maintenance of their cartilaginous phenotype. The hydrogel material effects on chondrogenic and osteogenic differentiation by BMSCs were evaluated in comparison to methacrylated gelatin hydrogel (GEL), a conventional bioink used for both chondrogenic and osteogenic applications. PGH and GEL hydrogels were loaded with goat BMSCs and cultured in chondrogenic and osteogenic mediums in vitro over six weeks. The PGH showed no sign of mineral deposition in an osteogenic environment in vitro. To further evaluate material effects, the hydrogels were loaded with adult human BMSCs (hBMSCs) and transforming growth factor ß-3 and grown in subcutaneous pockets in mice over eight weeks. Consistent with the in vitro results, the PGH had greater potential to induce chondrogenesis by BMSCs in vivo compared to the GEL as evidenced by elevated gene expression of chondrogenic markers, supporting its potential for stable cartilage engineering. The PGH also showed a greater percentage of GAG positive cells compared to the GEL. Unlike the GEL, the PGH hydrogel exhibited anti-osteogenic effects in vivo as evidenced by negative Von Kossa staining and suppressed gene expression of hypertrophic and osteogenic markers. By nature of their polymer composition alone, the PGH and GEL regulated BMSC differentiation down different osteochondral lineages. Thus, the PGH and GEL are promising hydrogels to regenerate stratified cartilaginous interfacial tissues in situ, such as the mandibular condyle surface, using undifferentiated BMSCs and a stratified scaffold design.

Bottom-Up Self-assembled Hydrogel-Mineral Composites Regenerate Rabbit Ulna Defect without Added Growth Factors.

Hydrogel-based biomaterials have advanced bone tissue engineering approaches in the last decade, through their ability to serve as a carrier for potent growth factor, bone morphogenic protein-2 (BMP-2). However, biophysical properties of hydrogels such as multiscale structural hierarchy and bone extracellular matrix (ECM)-mimetic microarchitecture are underutilized while designing current bone grafts. Incorporation of these properties offers great potential to create a favorable biomimetic microenvironment to harness their regenerative potential. Here, we present our approach to fabricate collagen-inspired bioactive hydrogel scaffolds (referred to as "RegenMatrix") to guide and enhance bone regeneration in a rabbit ulna defect model through the mimicry of multiscale architecture of bone ECM, i.e., native collagen. Specifically, we employed polyelectrolyte complexation to promote bottom-up self-assembly of oppositely charged polysaccharides (chitosan and kappa-carrageenan) at multiple length scales forming fibrils, which further assemble into fibers. The self-assembly and bioinspired scaffold fabrication method resulted in robust cylindrical RegenMatrix with excellent retention of the multiscale architecture and uniform mineral deposition throughout the scaffolds. RegenMatrix, in both nonmineralized and mineralized forms, enhanced bone regeneration in the semiload-bearing ulna defect when compared to the empty defect. RegenMatrix also showed greater histocompatibility without any fibrous tissue formation. Collectively, the RegenMatrix developed in this study has a great potential as a bioactive bone graft without any added growth factors.

Absence of Dipeptidyl Peptidase 3 Increases Oxidative Stress and Causes Bone Loss.

Controlling oxidative stress through the activation of antioxidant pathways is crucial in bone homeostasis, and impairments of the cellular defense systems involved contribute to the pathogenesis of common skeletal diseases. In this work we focused on the dipeptidyl peptidase 3 (DPP3), a poorly investigated ubiquitous zinc-dependent exopeptidase activating the Keap1-Nrf2 antioxidant pathway. We showed Dpp3 expression in bone and, to understand its role in this compartment, we generated a Dpp3 knockout (KO) mouse model and specifically investigated the skeletal phenotype. Adult Dpp3 KO mice showed a mild growth defect, a significant increase in bone marrow cellularity, and bone loss mainly caused by increased osteoclast activity. Overall, in the mouse model, lack of DPP3 resulted in sustained oxidative stress and in alterations of bone microenvironment favoring the osteoclast compared to the osteoblast lineage. Accordingly, in vitro studies revealed that Dpp3 KO osteoclasts had an inherent increased resorptive activity and ROS production, which on the other hand made them prone to apoptosis. Moreover, absence of DPP3 augmented bone loss after estrogen withdrawal in female mice, further supporting its relevance in the framework of bone pathophysiology. Overall, we show a nonredundant role for DPP3 in the maintenance of bone homeostasis and propose that DPP3 might represent a possible new osteoimmunological player and a marker of human bone loss pathology. © 2019 American Society for Bone and Mineral Research.

Preclinical Animal Models for Temporomandibular Joint Tissue Engineering.

There is a paucity of in vivo studies that investigate the safety and efficacy of temporomandibular joint (TMJ) tissue regeneration approaches, in part due to the lack of established animal models. Review of disease models for study of TMJ is presented herein with an attempt to identify relevant preclinical animal models for TMJ tissue engineering, with emphasis on the disc and condyle. Although degenerative joint disease models have been mainly performed on mice, rats, and rabbits, preclinical regeneration approaches must employ larger animal species. There remains controversy regarding the preferred choice of larger animal models between the farm pig, minipig, goat, sheep, and dog. The advantages of the pig and minipig include their well characterized anatomy, physiology, and tissue properties. The advantages of the sheep and goat are their easier surgical access, low cost per animal, and its high tissue availability. The advantage of the dog is that the joint space is confined, so migration of interpositional devices should be less likely. However, each species has limitations as well. For example, the farm pig has continuous growth until about 18 months of age, and difficult surgical access due to the zygomatic arch covering the lateral aspect of joint. The minipig is not widely available and somewhat costly. The sheep and the goat are herbivores, and their TMJs mainly function in translation. The dog is a carnivore, and the TMJ is a hinge joint that can only rotate. Although no species provides the gold standard for all preclinical TMJ tissue engineering approaches, the goat and sheep have emerged as the leading options, with the minipig as the choice when cost is less of a limitation; and with the dog and farm pig serving as acceptable alternatives. Finally, naturally occurring TMJ disorders in domestic species may be harnessed on a preclinical trial basis as a clinically relevant platform for translation.