Culture Conditions that Support Expansion and Chondrogenesis of Middle-Aged Rat Mesenchymal Stem Cells.

OBJECTIVE: Rats are an early preclinical model for cartilage tissue engineering, and a practical species for investigating the effects of aging. However, rats may be a poor aging model for mesenchymal stem cells (MSCs) based on laboratory reports of a severe decline in chondrogenesis beyond young adulthood. Such testing has not been conducted with MSCs seeded in a scaffold, which can improve the propensity of MSCs to undergo chondrogenesis. Therefore, the objective of this study was to evaluate chondrogenesis of middle-aged rat MSCs encapsulated in agarose. DESIGN: MSCs from 14- to 15-month-old rats were expanded, seeded into agarose, and cultured in chondrogenic medium with or without 5% serum for 15 days. Samples were evaluated for cell viability and cartilaginous extracellular matrix (ECM) accumulation. Experiments were repeated using MSCs from 6-week-old rats. RESULTS: During expansion, middle-aged rat MSCs demonstrated a diminishing proliferation rate that was improved ~2-fold in part by transient exposure to chondrogenic medium. In agarose culture in defined medium, middle-aged rat MSCs accumulated ECM to a much greater extent than negative controls. Serum supplementation improved cell survival ~2-fold, and increased ECM accumulation ~3-fold. Histological analysis indicated that defined medium supported chondrogenesis in a subset of cells, while serum-supplementation increased the frequency of chondrogenic cells. In contrast, young rat MSCs experienced robust chondrogenesis in defined medium that was not improved with serum-supplementation. CONCLUSIONS: These data demonstrate a previously-unreported propensity of middle-aged rat MSCs to undergo chondrogenesis, and the potential of serum to enhance chondrogenesis of aging MSCs.

Optimization of Degradation Profile for New Scaffold in Cartilage Repair.

Objective To establish whether a novel biomaterial scaffold with tunable degradation profile will aid in cartilage repair of chondral defects versus microfracture alone in vitro and in a rat model in vivo. Design In vitro-Short- and long-term degradation scaffolds were seeded with culture expanded articular chondrocytes or bone marrow mesenchymal stem cells. Cell growth and differentiation were evaluated with cell morphological studies and gene expression studies. In vivo-A microfracture rat model was used in this study to evaluate the repair of cartilage and subchondral bone with the contralateral knee serving as the empty control. The treatment groups include (1) empty osteochondral defect, (2) polycaprolactone copolymer-based polyester polyurethane-urea (PSPU-U) caffold short-term degradative profile, and (3) PSPU-U scaffold long-term degradative profile. After placement of the scaffold, the rats were then allowed unrestricted activity as tolerated, and histological analyses were performed at 4, 8, and 16 weeks. The cartilage defect was measured and compared with the contralateral control side. Results In vitro-Long-term scaffolds showed statistically significant higher levels of aggrecan and type II collagen expression compared with short-term scaffolds. In vivo-Within 16 weeks postimplantation, there was new subchondral bone formation in both scaffolds. Short-term scaffolds had a statistically significant increase in defect filling and better qualitative histologic fill compared to control. Conclusions The PSPU short-term degradation scaffold may aid in cartilage repair by ultimately incorporating the scaffold into the microfracture procedure.

Sonographic evaluation of knee cartilage defects implanted with preconditioned scaffolds.

OBJECTIVES: The purpose of this study was to develop a novel method for creating an acellular bioactive scaffold, to prove its efficacy in vivo and in vitro for the augmentation of biological repair, and to confirm that sonographic microscopy is a viable modality for monitoring the healing process of osteochondral defects implanted with preconditioned bioactive scaffolds. METHODS: Rabbit marrow stromal cells were retrovirally transduced with either bone morphogenetic protein 7 (BMP-7) or insulinlike growth factor 1 (IGF-1) genes, cultured for 9 weeks in nonwoven poly-L-lactic acid scaffolds, and then frozen and lyophilized. The knees were evaluated at 3, 6, and 12 weeks after surgery using 20-MHz ultrasound and then prepared for routine histologic analysis. B-scans of the extracellular matrix defects were compared to histologic results. RESULTS: Control defects showed a void or a mixture of fibrocartilage tissue. Both types of scaffolds resulted in a higher percentage (both P< .001) of primarily hyaline cartilage tissue with intact articular surfaces. The osteochondral defects were clearly observed in each sonographic signature. There were no differences between images of scaffolds treated with IGF-1 or BMP-7. Extracellular matrix regrowth was found to closely parallel (R(2) = 0.968; P < .003) the histologic images. A 3-mm defect depth and a 2.5-mm scaffold thickness were measured on the sonograms, comparing well to actual dimensions. CONCLUSIONS: There was a gradual increase in healing bordering the defects for the 3-, 6-, and 12-week samples. Also, we have shown that sonography can aid in monitoring implantation of preconditioned scaffolds in osteochondral defects and thus assessing the healing process and cartilage/bone quality.

Tissue Engineered Meniscus Repair: Influence of Cell Passage Number, Tissue Origin, and Biomaterial Carrier.

Objective. Studies have shown that meniscal repairs have better outcomes over both partial and total meniscectomies. Tissue engineering strategies to repair meniscus tears have been explored using cell sources that involve a donor as well as a period of in vitro cell expansion before use. This study explored cell sources that could be easily harvested and rapidly isolated by enzymatic digestion and cannulated delivery. Methods. Bovine menisci were used to create a bucket handle tear. Cell lines were established from meniscus, synovium, and adipose tissue and fluorescently labeled. At passages P2, P4, and P8, cells were added to the defect from the following experimental groups: cells alone, collagen gel, collagen scaffold, or hyaluronic acid. Menisci constructs were xenografted subcutaneously onto the dorsum of athymic rats and incubated for 3, 6, and 9 weeks, at which time they were retrieved and processed for histology. Results. Meniscal cells were able to repair defects faster and significantly better than adipose or synovium derived cells. Adipose cells were the least effective in comparison. Repair was significantly better at 9 weeks compared with 6 and 3 weeks. Macroscopic examination of menisci that received cell implants showed the thickest tissue in menisci that had collagen implants, and the thinnest fill occurred in menisci treated with cells alone. Histology confirmed no cells or integrative repair in the control specimens. Conclusions. Delivery of cells alone outperformed the additional use of biomaterials. Our results suggest a strategy that would use both meniscus and synovial cells for arthroscopic meniscal repair.

Articular Cartilage Repair: Where We Have Been, Where We Are Now, and Where We Are Headed.

This review traces the genealogy of the field of articular cartilage repair from its earliest attempts to its present day vast proliferation of research advances. Prior to the 1980s there was only sporadic efforts to regenerate articular cartilage as it was considered to be incapable of regeneration based on historical dogma. The first flurry of reports documented the use of various cell types ultimately leading to the first successful demonstration of autologous chondrocyte transplantation which was later translated to clinical use and has resulted in the revised axiom that cartilage regeneration is possible. The current field of cartilage repair is multifaceted and some of the 1980s' vintage concepts have been revisited with state of the art technology now available. The future of the field is now poised to undertake the repair of whole cartilage surfaces beyond focal defects and an appreciation for integrated whole joint health to restore cartilage homeostasis.

Tendon phenotype should dictate tissue engineering modality in tendon repair: a review.

Advancements in the technical aspects of tendon repair have significantly improved the treatment of tendon injuries. Arthroscopic techniques, suture material, and improved rehabilitation have all been contributing factors. Biological augmentation and tissue engineering appear to have the potential to improve clinical outcomes as well. After review of the physiology of tendon repair, three critical components of tissue engineering can be discerned: the cellular component, the carrier vehicle (matrix or scaffold), and the bioactive component (growth factors, platelet rich plasma). These three components are discussed with regard to each of three tendon types: Intra-synovial (flexor tendon), extra-synovial (Achilles tendon), and extra-synovial tendon under compression (rotator cuff). Scaffolds, biologically enhanced scaffolds, growth factors, platelet rich plasma, gene therapy, mesenchymal stem cells, and local environment factors in combination or alone may contribute to tendon healing. In the future it may be beneficial to differentiate these modes of healing augmentation with regard to tendon subtype.