Daniel J. Huey

Unlike Bone, Cartilage Regeneration Remains Elusive

Articular cartilage was predicted to be one of the first tissues to successfully be regenerated, but this proved incorrect. In contrast, bone (but also vasculature and cardiac tissues) has seen numerous successful reparative approaches, despite consisting of multiple cell and tissue types and, thus, possessing more complex design requirements. Here, we use bone-regeneration successes to highlight cartilage-regeneration challenges: such as selecting appropriate cell sources and scaffolds, creating biomechanically suitable tissues, and integrating to native tissue. We also discuss technologies that can address the hurdles of engineering a tissue possessing mechanical properties that are unmatched in human-made materials and functioning in environments unfavorable to neotissue growth.

Maturational growth of self-assembled, functional menisci as a result of TGF-β1 and enzymatic chondroitinase-ABC stimulation

Replacement of the knee meniscus requires a material possessing adequate geometrical and biomechanical properties. Meniscal tissue engineering attempts have been unable to produce tissue with collagen content and biomechanical properties, particularly tensile properties, mimicking native menisci. In an effort to obtain the geometric properties and the maturational growth necessary for the recapitulation of biochemical and, thus, biomechanical properties, a scaffoldless cell-based system, the self-assembly process, was used in conjunction with the catabolic enzyme chondroitinase-ABC and TGF-β1. We show that combinations of these agents resulted in maturational growth as evidenced by synergistic enhancement of the radial tensile modulus by 5-fold and the compressive relaxation modulus by 68%, and additive increases of the compressive instantaneous modulus by 136% and Col/WW by 196%. This study shows that tissue engineering can produce a biomaterial that is on par with the biochemical and biomechanical properties of native menisci.

Tension-compression loading with chemical stimulation results in additive increases to functional properties of anatomic meniscal constructs.

Objective This study aimed to improve the functional properties of anatomically-shaped meniscus constructs through simultaneous tension and compression mechanical stimulation in conjunction with chemical stimulation. Methods Scaffoldless meniscal constructs were subjected to simultaneous tension and compressive stimulation and chemical stimulation. The temporal aspect of mechanical loadingwas studied by employing two separate five day stimulation periods. Chemical stimulation consisted of the application of a catabolic GAG-depleting enzyme, chondroitinase ABC (C-ABC), and an anabolic growth factor, TGF-β1. Mechanical and chemical stimulation combinations were studied through a full-factorial experimental design and assessed for histological, biochemical, and biomechanical properties following 4 wks of culture. Results Mechanical loading applied from days 10–14 resulted in significant increases in compressive, tensile, and biochemical properties of meniscal constructs. When mechanical and chemical stimuliwere combined significant additive increases in collagen per wet weight (4-fold), compressive instantaneous (3-fold) and relaxation (2-fold) moduli, and tensile moduli in the circumferential (4-fold) and radial (6-fold) directions were obtained. Conclusions This study demonstrates that a stimulation regimen of simultaneous tension and compression mechanical stimulation, C-ABC, and TGF-β1 is able to create anatomic meniscus constructs replicating the compressive mechanical properties, and collagen and GAG content of native tissue. In addition, this study significantly advances meniscus tissue engineering by being the first to apply simultaneous tension and compression mechanical stimulation and observe enhancement of tensile and compressive properties following mechanical stimulation.

Regenerating Mandibular Bone Using rhBMP-2: Part 1— Immediate Reconstruction of Segmental Mandibulectomies

OBJECTIVE To describe a surgical technique using a regenerative approach and internal fixation for immediate reconstruction of critical size bone defects after segmental mandibulectomy in dogs. STUDY DESIGN Prospective case series. ANIMALS Dogs (n = 4) that had reconstruction after segmental mandibulectomy for treatment of malignant or benign tumors. METHODS Using a combination of extraoral and intraoral approaches, a locking titanium plate was contoured to match the native mandible. After segmental mandibulectomy, the plate was secured and a compression resistant matrix (CRM) infused with rhBMP-2, implanted in the defect. The implant was then covered with a soft tissue envelope followed by intraoral and extraoral closure. RESULTS All dogs that had mandibular reconstruction healed with intact gingival covering over the mandibular defect and had immediate return to normal function and occlusion. Mineralized tissue formation was observed clinically within 2 weeks and solid cortical bone formation within 3 months. CT findings at 3 months showed that the newly regenerated mandibular bone had ∼50% of the bone density and porosity compared to the contralateral side. No significant complications occurred. CONCLUSION Mandibular reconstruction using internal fixation and CRM infused with rhBMP-2 is an excellent solution for immediate reconstruction of segmental mandibulectomy defects in dogs.

Enhancing Post-Expansion Chondrogenic Potential of Costochondral Cells in Self-Assembled Neocartilage

The insufficient healing capacity of articular cartilage necessitates mechanically functional biologic tissue replacements. Using cells to form biomimetic cartilage implants is met with the challenges of cell scarcity and donor site morbidity, requiring expanded cells that possess the ability to generate robust neocartilage. To address this, this study assesses the effects of expansion medium supplementation (bFGF, TFP, FBS) and self-assembled construct seeding density (2, 3, 4 million cells/5 mm dia. construct) on the ability of costochondral cells to generate biochemically and biomechanically robust neocartilage. Results show TFP (1 ng/mL TGF-β1, 5 ng/mL bFGF, 10 ng/mL PDGF) supplementation of serum-free chondrogenic expansion medium enhances the post-expansion chondrogenic potential of costochondral cells, evidenced by increased glycosaminoglycan content, decreased type I/II collagen ratio, and enhanced compressive properties. Low density (2 million cells/construct) enhances matrix synthesis and tensile and compressive mechanical properties. Combined, TFP and Low density interact to further enhance construct properties. That is, with TFP, Low density increases type II collagen content by over 100%, tensile stiffness by over 300%, and compressive moduli by over 140%, compared with High density. In conclusion, the interaction of TFP and Low density seeding enhances construct material properties, allowing for a mechanically functional, biomimetic cartilage to be formed using clinically relevant costochondral cells.

A proposed model of naturally occurring osteoarthritis in the domestic rabbit.

Osteoarthritis affects one in eight American adults over the age of 25 y and is a leading cause of chronic disability in the US. Translational research to investigate treatments for this naturally occurring joint disease requires an appropriate animal model. The authors conducted a retrospective study to assess the potential of naturally occurring osteoarthritis in the domestic rabbit as a model of the human disease. Analysis of radiographic images showed that the presence and severity of osteoarthritis were significantly influenced by both age and body weight. The most commonly affected joints were the knee and the hip. The findings reported here suggest that the rabbit is an excellent model of spontaneously arising osteoarthritis that may be useful in translational research pertaining to the human disease.

Alteration of the Fibrocartilaginous Nature of Scaffoldless Constructs Formed from Leporine Meniscus Cells and Chondrocytes through Manipulation of Culture and Processing Conditions

Articular cartilage and the menisci of the knee joint lack intrinsic repair capacity; thus, injuries to these tissues result in eventual osteoarthrotic changes to the joint. Tissue engineering offers the potential to replace damaged cartilage and mitigate long-term debilitating changes to the joint. In an attempt to enhance the ability of adult articular chondrocytes (ACs) and meniscus cells (MCs) to produce robust scaffoldless neocartilage, the effects of passage number, cryopreservation, and redifferentiation prior to construct formation were studied. By increasing passage number, smaller donor biopsies could be used to generate sufficient cells for tissue engineering and, in this study, no detrimental effects were observed when employing passage-4 versus passage-3 cells. Cryopreservation of cells would enable the generation of a cell bank thus reducing lead time and enhancing consistency of cell-based therapies. Interestingly, cryopreservation was shown to enhance the biomechanical properties of the resultant self-assembled constructs. With regard to redifferentiation prior to construct formation, aggregate redifferentiation was shown to enhance the biochemical and biomechanical properties of self-assembled constructs. By increasing passaging number, cryopreserving cells, and applying aggregate redifferentiation prior to neotissue formation, the utility of ACs and MCs in tissue engineering can be enhanced.

Chondrogenically Tuned Expansion Enhances the Cartilaginous Matrix-Forming Capabilities of Primary, Adult, Leporine Chondrocytes

When expanded through passage, chondrocytes lose their ability to produce high-quality cartilaginous matrix. This study attempts to improve the properties of constructs formed with expanded chondrocytes through alterations in the expansion protocol and the ratio of primary to expanded chondrocytes used to form cartilage constructs. A chondrogenically tuned expansion protocol provided similar monolayer growth rates as those obtained using serum-containing medium and enhanced cartilaginous properties of resultant constructs. Various ratios of primary to chondrogenically expanded chondrocytes were then self-assembled to form neocartilage. Biochemical analysis showed that constructs formed with only expanded cells had twice the GAG per wet weight and collagen II/collagen I ratio compared to constructs formed with primary chondrocytes. Biomechanically, compressive properties of constructs formed with only passaged cells matched the instantaneous modulus and exceeded the relaxation modulus of constructs formed with only primary cells. These counterintuitive results show that, by applying proper expansion and three-dimensional culture techniques, the cartilage-forming potential of adult chondrocytes expanded through passage can be enhanced over that of primary cells.

Regenerating Mandibular Bone Using rhBMP-2: Part 2—Treatment of Chronic, Defect Non-Union Fractures

OBJECTIVE To describe a surgical technique using a regenerative approach and internal fixation for reconstruction of critical size bone defect non-union mandibular fractures. STUDY DESIGN Case series. ANIMALS Dogs (n = 6) that had internal fixation of defect non-union mandibular fracture. METHODS In 5 dogs, the repair was staged and extraction of teeth performed during the initial procedure. After 21-98 days (mean, 27 days) pharyngotomy intubation and temporary maxillomandibular fixation were performed. Using an extraoral approach, a locking titanium miniplate was contoured and secured to the mandible. A compression resistant matrix (CRM) infused with rhBMP-2 was implanted in the defect. The implant was then covered with a soft tissue envelope followed by surgical wound closure. RESULTS All dogs healed with intact gingival covering over the mandibular fracture site defect and had immediate return to normal function and correct occlusion. Hard-tissue formation was observed clinically within 2 weeks and solid cortical bone formation within 3 months. CT findings in 1 dog at 3 months postoperatively demonstrated that the newly regenerated mandibular bone had 92% of the bone density and porosity compared to the contralateral side. Long-term follow-up revealed excellent outcome. CONCLUSION Mandibular reconstruction using internal fixation and CRM infused with rhBMP-2 is an excellent solution for the treatment of critical size defect non-union fractures in dogs.

The temporomandibular joint disc of Asian elephant (Elephas maximus) and African elephant (Loxodonta africana)

The temporomandibular joint (TMJ) is a synovial articulation between the mandibular head of the condylar process of the mandible and the mandibular fossa of the squamous temporal bone. Extensions of the fibrous capsule into the joint space form a biconcave disc that functions as an articulating surface and divides the joint into dorsal and ventral compartments. The TMJ disc plays a crucial role in normal functioning of the joint, and differences in cranial morphology, mastication patterns, and diet are reflected in the material and biochemical properties of the disc. The purpose of the present case study was to compare the regional histologic differences between two elephant genera and quantify the biochemical and biomechanical properties of the African elephant disc. This study provides a unique insight into the elephant TMJ disc and also provides a comparison between the African and the Asian elephant genera. The results demonstrate several remarkable findings. First, structure–function relationships exist within the elephant TMJ disc. Second, regional variations exist in the elephant TMJ disc, and these are likely to correlate with its functional requirement. Additionally, it is apparent that some properties of the disc vary with the specific anatomy, diet requirement, and jaw motion. Finally, in comparison with the TMJ disc of other species, it is clear that, although the elephant disc is unique, it has properties that transcend and are preserved among the species.