Alejandro J. Almarza

Design characteristics for the tissue engineering of cartilaginous tissues.

Tissues like the temporomandibular joint (TMJ) disc and the knee meniscus are often mistakenly viewed as a tantamount to hyaline cartilage, largely due to the absence of a comprehensive understanding of the distinguishing properties of cartilaginous tissues. Because of this confusion, fibrocartilaginous tissue engineering attempts may not be based on suitable experimental designs. Fibrocartilaginous tissues are markedly different than hyaline cartilage; however, the dearth of knowledge related to their cellular and biochemical composition, as well as their bio- mechanical characteristics, is stunning. Hyaline articular cartilage is exclusively composed of chondrocytes that produce primarily type II collagen, whereas the TMJ disc and the knee meniscus have a mixed cell population of fibroblasts and cells similar to chondrocytes, which predominantly secrete type I collagen. Additionally, fibrocartilaginous tissues have a low glycosaminoglycan content, a low compressive modulus, and a high tensile modulus when compared to hyaline cartilage. Therefore, it is crucial for fibrocartilaginous tissue engineering attempts to be tissue-specific, utilizing the knowledge of the distinct and unique properties of these tissues. At the same time, advances and insights related to the science and engineering aspect of hyaline cartilage regeneration must be carefully considered for the in vitro engineering of fibrocartilaginous tissues.

Effects of initial cell seeding density for the tissue engineering of the temporomandibular joint disc

Tissue engineering may provide a better treatment modality for postoperative discectomy patients. The TMJ disc is an ideal candidate for tissue engineering approaches because of its lack of an intrinsic regenerative ability. Unfortunately, basic knowledge related to TMJ disc tissue engineering is still at an infancy level and not on par to that related to articular cartilage tissue engineering. The objective of this study was to examine the effects of initial cell density of TMJ disc cells seeded in nonwoven poly-glycolic acid (PGA) scaffolds on the biochemical and biomechanical properties of constructs examined at 0, 3, and 6 weeks after seeding. Low, medium, and high seeding densities were chosen to be 15, 30, and 120 million cells per ml of scaffold, which were seeded using a spinner flask. Significant differences were found temporally and as a function of seeding density in morphology, total collagen, GAG content, and permeability of the constructs, but not in aggregate modulus. The high seeding density group outperformed the low and medium groups in collagen and GAG content at all time points measured. The high-density group produced a total of 55.37 ± 3.56 μg of collagen per construct, maintained 15.77 ± 1.86 μg of GAG per construct, and only shrunk to 50% of the original scaffold size. Permeability of the constructs at 6 weeks was decreased by 70% compared to 0 weeks.

Magnesium alloys as a biomaterial for degradable craniofacial screws.

Recently, magnesium (Mg) alloys have received significant attention as potential biomaterials for degradable implants, and this study was directed at evaluating the suitability of Mg for craniofacial bone screws. The objective was to implant screws fabricated from commercially available pure Mg and alloy AZ31 in vivo in a rabbit mandible. First, Mg and AZ31 screws were compared to stainless steel screws in an in vitro pull-out test and determined to have a similar holding strength (∼40N). A finite-element model of the screw was created using the pull-out test data, and this model can be used for future Mg alloy screw design. Then, Mg and AZ31 screws were implanted for 4, 8 and 12weeks, with two controls of an osteotomy site (hole) with no implant and a stainless steel screw implanted for 12weeks. Microcomputed tomography was used to assess bone remodeling and Mg/AZ31 degradation, both visually and qualitatively through volume fraction measurements for all time points. Histological analysis was also completed for the Mg and AZ31 at 12weeks. The results showed that craniofacial bone remodeling occurred around both Mg and AZ31 screws. Pure Mg had a different degradation profile than AZ31; however, bone growth occurred around both screw types. The degradation rate of both Mg and AZ31 screws in the bone marrow space and the muscle were faster than in the cortical bone space at 12weeks. Furthermore, it was shown that by alloying Mg, the degradation profile could be changed. These results indicate the promise of using Mg alloys for craniofacial applications.

Effects of a bioscaffold on collagen fibrillogenesis in healing medial collateral ligament in rabbits.

Bioscaffolds have been successfully used to improve the healing of ligaments and tendons. In a rabbit model, the application of porcine small intestine submucosa (SIS) to the healing medial collateral ligament (MCL) resulted in improved mechanical properties with the formation of larger collagen fibrils. Thus, the objective of the study was to find out whether the SIS bioscaffold could improve the gene expressions of fibrillogenesis‐related molecules, specifically, collagen types I, III, V, and small leucine‐rich proteoglycans including decorin, biglycan, lumican, and fibromodulin, as well as collagen fibril morphology and organization, in the healing rabbit MCL at an early time point (6 weeks postinjury). Twenty skeletally mature rabbits were equally divided into two groups. In the SIS‐treated group, a 6‐mm gap was surgically created and a layer of SIS was sutured to cover the gap, whereas the gap was left open in the nontreated group. At 6 weeks postinjury, Massons trichrome staining showed that the SIS‐treated group had more regularly aligned collagen fibers and cells. Transmission electron microscopy revealed that the SIS‐treated group had larger collagen fibrils with a diameter distribution from 24 to 120 nm, whereas the nontreated group had only small collagen fibrils (ranging from 26 to 87 nm, p < 0.05). Finally, the quantitative real‐time PCR showed that the mRNAs of collagen type V, decorin, biglycan, and lumican in the SIS‐treated group were 41, 58, 51, and 43% lower than those in the nontreated group, respectively (p < 0.05). Such significant reduction in the gene expressions are closely related to the improved morphological characteristics, which are known to be coupled with better mechanical properties, as previously reported in longer term studies.

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.

Poly (glycerol sebacate): a novel scaffold material for temporomandibular joint disc engineering.

The preponderance of temporomandibular joint (TMJ) disorders involving TMJ disc injury inspires the need to further explore tissue engineering strategies. The objective of this study was to examine the potential of poly (glycerol sebacate) (PGS), a biocompatible, biodegradable elastomer, as a porous scaffold material for the TMJ disc. Goat fibrochondrocytes were seeded on PGS at three seeding densities (25, 50, 100 million cells/mL scaffold), respectively, and cultured for 24 h, 2 weeks, and 4 weeks. The resulting histological, biochemical, and biomechanical properties were determined. Histological staining revealed an abundance of both collagen and glycosaminoglycans (GAG) throughout the high seeding density scaffolds at 4 weeks. There was also a significant increase in the cellular content in all groups over the four-week period, showing that the scaffolds promoted cell attachment and proliferation. The PGS scaffolds supported the deposition of large quantities of extracellular matrix, with differences noted between seeding density groups. At 4 weeks, the medium and high seeding density groups had significantly more collagen per scaffold (181±46 μg and 218±24 μg, respectively) than the low seeding density group (105±28 μg) (p<0.001). At 4 weeks, the medium and high seeding density groups also had a significantly higher GAG content per scaffold (702±253 μg and 773±187 μg, respectively), than the low seeding density group (324±73 μg) (p<0.001). The compression tangent modulus was significantly greater at 4 weeks than 24 h (123.6±86 kPa and 26.2±5 kPa, respectively) (seeding density groups combined) (p<0.001), with no differences between seeding groups at each time point. After 4 weeks, the tangent modulus of the low seeding density group was in a similar range of the goat TMJ disc (180±127 kPa compared to 304±141 kPa, respectively). The results show that cell seeding density and culture time do have an effect on both the biochemical and biomechanical properties of PGS scaffolds. These findings demonstrate that PGS has great potential as a scaffold material for TMJ disc engineering.

Effects of Ascorbic Acid Concentration on the Tissue Engineering of the Temporomandibular Joint Disc

Abstract The temporomandibular joint (TMJ) disc is a specialized fibrocartilaginous tissue. When the disc becomes an obstacle and becomes damaged, surgeons have no choice but to perform a discectomy. Tissue engineering may provide a novel treatment modality for TMJ disorder patients who undergo discectomy. No studies have been conducted on the most favourable media for TMJ disc cells. The objective of the current study was to examine the effects on biochemical and biomechanical properties of varying ascorbic acid concentrations (0, 25, or 50 μg/ml) on TMJ disc cells seeded on non-woven PGA scaffolds. The ascorbic acid concentration of the 25 μg/ml group resulted in more effective cell seeding of the scaffolds, with 1.53 million cells per construct, by comparison with the 0 and 50 μg/ml groups which had 1.20 million and 1.32 million cells per scaffold respectively. At week 4, the 25 μg/ml group had a higher collagen content than the 0 μg/ml group, with 30.4 ± 2.7 and 24.9 ± 3.3 μg of collagen per construct respectively. The 25 μg/ml group had a higher aggregate modulus than the 50 μg/ml group, with values of 6.1 ± 1.3 and 4.0 ± 0.9 kPa respectively at week 4. The results of this study indicate that the use of 25 μg/ml of ascorbic acid in culture media is effective for the tissue engineering of the TMJ disc, significantly outperforming media without or with 50 μg/ml of ascorbic acid.

Tissue Engineering of Temporomandibular Joint Cartilage

The temporomandibular joint (TMJ) is a site of intense morbidity for millions of people, especially young, pre-menopausal women. Central to TMJ afflictions are the cartilaginous tissues of the TMJ, especially those of the disc and condylar cartilage, which play crucial roles in normal function of this unusual joint. Damage or disease to these tissues significantly impacts a patients quality of life by making common activities such as talking and eating difficult and painful. Unfortunately, these tissues have limited ability to heal, necessitating the development of treatments for repair or replacement. The burgeoning field of tissue engineering holds promise that replacement tissues can be constructed in the laboratory to recapitulate the functional requirements of native tissues. This book outlines the biomechanical, biochemical, and anatomical characteristics of the disc and condylar cartilage, and also provides a historical perspective of past and current TMJ treatments and previ us tissue engineering efforts. This book was written to serve as a reference for researchers seeking to learn about the TMJ, for undergraduate and graduate level courses, and as a compendium of TMJ tissue engineering design criteria. Table of Contents: The Temporomandibular Joint / Fibrocartilage of the TMJ Disc / Cartilage of the Mandibular Condyle / Tissue Engineering of the Disc / Tissue Engineering of the Mandibular Condyle / Current Perspectives

Poly(glycerol sebacate) elastomer: a novel material for mechanically loaded bone regeneration.

The selection criteria for potential bone engineering scaffolds are based chiefly on their relative mechanical comparability to mature bone. In this study, we challenge this notion by obtaining full regeneration of a rabbit ulna critical size defect by employing the elastomeric polymer, poly(glycerol sebacate) (PGS). We tested the regeneration facilitated by PGS alone, PGS in combination with hydroxyapatite particles, or PGS seeded with bone marrow stromal cells. We investigated the quantity and quality of the regenerated bone histologically, by microcomputed tomography and by four-point bending flexural mechanical testing at 8 weeks postimplantation. We conclude that the relatively lower stiffness of this biocompatible elastomer allows a load-transducing milieu in which osteogenesis, matrix deposition, and eventual bone maturation can take place. This studys results suggest that PGS elastomer is an auspicious osteoconductive material for the regeneration of bony defects. These results call for an innovative reassessment of the current art of selection for novel bone scaffold materials.

A comparison of the mechanical properties of the goat temporomandibular joint disc to the mandibular condylar cartilage in unconfined compression.

The aim of this study was to make a comparison of the compressive properties of the goat temporomandibular joint (TMJ) disc to the mandibular condylar cartilage (MCC) and to explore the transversely isotropic biphasic model. Samples taken mediolaterally from three regions of the TMJ disc and MCC were tested in unconfined compression at strain levels ranging from 10% to 50% and then assessed for biochemical content. The results indicated that the TMJ disc exhibits a significantly greater tangent modulus than the MCC from 20% to 50% strain with values ranging from 729 ± 267 to 2413 ± 406 kPa and 363 ± 169 to 1677 ± 538 kPa, respectively (P < .05). The collagen content of the TMJ disc was significantly greater than the MCC, while the opposite held for the glycosaminoglycan (GAG) and DNA content. The results emphasize fundamental differences between the articulating tissues of the TMJ.