Luis A. Solchaga

The Chondrogenic Potential of Human Bone-marrow-derived Mesenchymal Progenitor Cells*

Mesenchymal progenitor cells provide a source of cells for the repair of musculoskeletal tissue. However, in vitro models are needed to study the mechanisms of differentiation of progenitor cells. This study demonstrated the successful induction of in vitro chondrogenesis with human bone-marrow-derived osteochondral progenitor cells in a reliable and reproducible culture system. Human bone marrow was removed and fractionated, and adherent cell cultures were established. The cells were then passaged into an aggregate culture system in a serum-free medium. Initially, the cell aggregates contained type-I collagen and neither type-II nor type-X collagen was detected. Type-II collagen was typically detected in the matrix by the fifth day, with the immunoreactivity localized in the region of metachromatic staining. By the fourteenth day, type-II and type-X collagen were detected throughout the cell aggregates, except for an outer region of flattened, perichondrial-like cells in a matrix rich in type-I collagen. Aggrecan and link protein were detected in extracts of the cell aggregates, providing evidence that large aggregating proteoglycans of the type found in cartilaginous tissues had been synthesized by the newly differentiating chondrocytic cells; the small proteoglycans, biglycan and decorin, were also detected in extracts. Immunohistochemical staining with antibodies specific for chondroitin 4-sulfate and keratan sulfate demonstrated a uniform distribution of proteoglycans throughout the extracellular matrix of the cell aggregates. When the bone-marrow-derived cell preparations were passaged in monolayer culture as many as twenty times, with cells allowed to grow to confluence at each passage, the chondrogenic potential of the cells was maintained after each passage. CLINICAL RELEVANCE: Chondrogenesis of progenitor cells is the foundation for the in vivo repair of fractures and damaged articular cartilage. In vitro chondrogenesis of human bone-marrow-derived osteochondral progenitor cells should provide a useful model for studying this cellular differentiation. Furthermore, the maintenance of chondrogenic potential after greater than a billion-fold expansion provides evidence for the clinical utility of these cells in the repair of bone and cartilage.

FGF‐2 enhances the mitotic and chondrogenic potentials of human adult bone marrow‐derived mesenchymal stem cells

Human mesenchymal stem cells (hMSCs) expanded with and without fibroblast growth factor (FGF) supplementation were compared with respect to their proliferation rate, ability to differentiate along the chondrogenic pathway in vitro, and their gene expression profiles. hMSCs expanded in FGF‐supplemented medium were smaller and proliferated more rapidly than hMSCs expanded in control conditions. Chondrogenic cultures made with FGF‐treated cells were larger and contain more proteoglycan than those made with control cells. Furthermore, aggregates of FGF‐treated cells lacked the collagen type I‐positive and collagen type II‐negative outer layer characteristic of aggregates of control cells. A total of 358 unique transcripts were differentially expressed in FGF‐treated hMSCs. Of these, 150 were upregulated and 208 downregulated. Seventeen percent of these genes affect proliferation. Known genes associated with cellular signaling functions comprised the largest percentage (∼20%) of differentially expressed transcripts. Eighty percent of differentially expressed extracellular matrix‐related genes were downregulated. The present findings that FGF‐2 enhances proliferation and differentiation of hMSCs adds to a growing body of evidence that cytokines modulate the differentiation potential and, perhaps, the multipotentiality of adult stem cells. With the generation of gene expression profiles of FGF‐treated and control cells we have taken the first steps in the elucidation of the molecular mechanism(s) behind these phenomena.

Tissue-Engineered Fabrication of an Osteochondral Composite Graft Using Rat Bone Marrow-Derived Mesenchymal Stem Cells

This study tested the tissue engineering hypothesis that construction of an osteochondral composite graft could be accomplished using multipotent progenitor cells and phenotype-specific biomaterials. Rat bone marrow-derived mesenchymal stem cells (MSCs) were culture-expanded and separately stimulated with transforming growth factor-beta1 (TGF-beta1) for chondrogenic differentiation or with an osteogenic supplement (OS). MSCs exposed to TGF-beta1 were loaded into a sponge composed of a hyaluronan derivative (HYAF-11) for the construction of the cartilage component of the composite graft, and MSCs exposed to OS were loaded into a porous calcium phosphate ceramic component for bone formation. Cell-loaded HYAFF-11 sponge and ceramic were joined together with fibrin sealant, Tisseel, to form a composite osteochondral graft, which was then implanted into a subcutaneous pocket in syngeneic rats. Specimens were harvested at 3 and 6 weeks after implantation, examined with histology for morphologic features, and stained immunohistochemically for type I, II, and X collagen. The two-component composite graft remained as an integrated unit after in vivo implantation and histologic processing. Fibrocartilage was observed in the sponge, and bone was detected in the ceramic component. Observations with polarized light indicated continuity of collagen fibers between the ceramic and HYAFF-11 components in the 6-week specimens. Type I collagen was identified in the neo-tissue in both sponge and ceramic, and type II collagen in the fibrocartilage, especially the pericellular matrix of cells in the sponge. These data suggest that the construction of a tissue-engineered composite osteochondral graft is possible with MSCs and different biomaterials and bioactive factors that support either chondrogenic or osteogenic differentiation.

Bioreactors mediate the effectiveness of tissue engineering scaffolds

We hypothesized that the mechanically active environment present in rotating bioreactors mediates the effectiveness of three‐dimensional (3D) scaffolds for cartilage tissue engineering. Cartilaginous constructs were engineered by using bovine calf chondrocytes in conjunction with two scaffold materials (SM) (benzylated hyaluronan and polyglycolic acid); three scaffold structures (SS) (sponge, non‐woven mesh, and composite woven/non‐woven mesh); and two culture systems (CS) (a bioreactor system and petri dishes). Construct size, composition [cells, glycosaminoglycans (GAG), total collagen, and type‐specific collagen mRNA expression and protein levels], and mechanical function (compressive modulus) were assessed, and individual and interactive effects of model system parameters (SM, SS, CS, SM∗CS and SS∗CS) were demonstrated. The CS affected cell seeding (higher yields of more spatially uniform cells were obtained in bioreactor‐grown than dish‐grown 3‐day constructs) and subsequently affected chondrogenesis (higher cell numbers, wet weights, wet weight GAG fractions, and collagen type II levels were obtained in bioreactor‐grown than dish‐grown 1‐month constructs). In bioreactors, mesh‐based scaffolds yielded 1‐month constructs with lower type I collagen levels and four‐fold higher compressive moduli than corresponding sponge‐based scaffolds. The data imply that interactions between bioreactors and 3D tissue engineering scaffolds can be utilized to improve the structure, function, and molecular properties of in vitro‐generated cartilage.

A novel serum-free medium for the expansion of human mesenchymal stem cells.

IntroductionHuman multipotent mesenchymal stem cell (MSC) therapies are being tested clinically for a variety of disorders, including Crohns disease, multiple sclerosis, graft-versus-host disease, type 1 diabetes, bone fractures, and cartilage defects. However, despite the remarkable clinical advancements in this field, most applications still use traditional culture media containing fetal bovine serum. The ill-defined and highly variable nature of traditional culture media remains a challenge, hampering both the basic and clinical human MSC research fields. To date, no reliable serum-free medium for human MSCs has been available.MethodsIn this study, we developed and tested a serum-free growth medium on human bone marrow-derived MSCs through the investigation of multiple parameters including primary cell isolation, multipassage expansion, mesoderm differentiation, cellular phenotype, and gene-expression analysis.ResultsSimilar to that achieved with traditional culture medium, human MSCs expanded in serum-free medium supplemented with recombinant human platelet-derived growth factor-BB (PDGF-BB), basic fibroblast growth factor (bFGF), and transforming growth factor (TGF)-β1 showed extensive propagation with retained phenotypic, differentiation, and colony-forming unit potential. To monitor global gene expression, the transcriptomes of bone marrow-derived MSCs expanded under serum-free and serum-containing conditions were compared, revealing similar expression profiles. In addition, the described serum-free culture medium supported the isolation of human MSCs from primary human marrow aspirate with continual propagation.ConclusionsAlthough the described serum-free MSC culture medium is not free of xenogeneic components, this medium provides a substitute for serum-containing medium for research applications, setting the stage for future clinical applications.

CHONDROPROGENITOR CELLS OF SYNOVIAL TISSUE

OBJECTIVE To assess the chondrogenic potential of cells within the synovium. METHODS Explants of synovium taken from various sites in the joint were embedded in agarose and cultured with transforming growth factor beta1 (TGFbeta1) to assess their chondrogenic potential. Isolated synovial cells were also tested for their chondrogenic potential by culturing them as aggregates in a chemically defined medium with TGFbeta1. Cartilage formation was determined with histologic staining and immunohistochemistry. The osteochondral potential of the isolated cells was also assessed after subcutaneous implantation of the cells, loaded into porous calcium phosphate ceramic cubes, in athymic mice. RESULTS A total of 48 synovial explants were cultured in agarose with TGFbeta1. The formation of cartilage was observed in the outer region of 21 explants, and type II collagen was localized in that region by immunohistochemistry. A larger percentage of TGFbeta1+ explants from the inner synovium sites formed cartilage compared with those from the outer synovium sites. Chondrogenesis occurred in aggregates incubated with TGFbeta1 as early as day 7, and by day 14, all TGFbeta1+ aggregates demonstrated chondrogenesis. In contrast with the results of the in vitro aggregate assay for chondrogenesis, no formation of cartilage or bone was evident in any section containing synovial cell-loaded ceramic cubes that were harvested at either 3 or 6 weeks after implantation subcutaneously in athymic mice. CONCLUSION Synovial explants and isolated synovial cells will undergo chondrogenesis when cultured in the presence of TGFbeta1. The data indicate a possible synovial origin for the chondrocytic cells found in rheumatoid pannus. Furthermore, these data are consistent with the clinical findings of synovial chondrogenesis leading to synovial chondromatosis.

Treatment of Osteochondral Defects with Autologous Bone Marrow in a Hyaluronan-Based Delivery Vehicle

The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable and bioactive materials that provide structural support and molecular cuing to stimulate repair. Since bone marrow contains osteochondral progenitor cells and bioactive agents, it is hypothesized that the combination of scaffold and bone marrow would be a superior composite material for osteochondral repair. This hypothesis will be tested by comparing the outcome of osteochondral defects filled with a fibronectin-coated hyaluronan-based sponge (ACP) with or without autologous bone marrow. Thirty-three 4-month-old rabbits received 3-mm diameter osteochondral defects that were then filled with ACP loaded or not with autologous bone marrow. Rabbits were sacrificed at 2, 3, 4, 12, and 24 weeks after surgery and the condyles processed for histologic and immunohistochemical evaluation. The defects were graded with a histologic scoring scale. Except for the 3-week specimens, the histologic appearance of the defects was similar in both groups. Four weeks after surgery, the defects were filled with bone with a top layer of cartilage well integrated with the adjacent cartilage. Twelve and 24 weeks after surgery, the defects again showed bone filling. The primary difference between the 4-week samples and the 12- and 24-week samples was that the layer of cartilage that appeared to be thinner than the adjacent cartilage. At each harvest time, the overall histologic scores of the specimens did not reveal statistical differences between the treatment groups. However, as revealed by the results of the 3-week sacrifices, bone marrow loading appeared to accelerate the first stages of the repair process. The fibronectin-coated hyaluronan-based scaffold appears to organize the natural response and facilitate the integration of the neo-cartilage with the adjacent tissue. The fundamental tissue engineering principles derived from this study should provide guidelines for the development of comparable clinical reconstructive therapies.

BMP‐2 induction and TGF‐β1 modulation of rat periosteal cell chondrogenesis

Periosteum contains osteochondral progenitor cells that can differentiate into osteoblasts and chondrocytes during normal bone growth and fracture healing. TGF‐β1 and BMP‐2 have been implicated in the regulation of the chondrogenic differentiation of these cells, but their roles are not fully defined. This study was undertaken to investigate the chondrogenic effects of TGF‐β1 and BMP‐2 on rat periosteum‐derived cells during in vitro chondrogenesis in a three‐dimensional aggregate culture. RT‐PCR analyses for gene expression of cartilage‐specific matrix proteins revealed that treatment with BMP‐2 alone and combined treatment with TGF‐β1 and BMP‐2 induced time‐dependent mRNA expression of aggrecan core protein and type II collagen. At later times in culture, the aggregates treated with BMP‐2 exhibited expression of type X collagen and osteocalcin mRNA, which are markers of chondrocyte hypertrophy. Aggregates incubated with both TGF‐β1 and BMP‐2 showed no such expression. Treatment with TGF‐β1 alone did not lead to the expression of type II or X collagen mRNA, indicating that this factor itself did not independently induce chondrogenesis in rat periosteal cells. These data were consistent with histological and immunohistochemical results. After 14 days in culture, BMP‐2‐treated aggregates consisted of many hypertrophic chondrocytes within a metachromatic matrix, which was immunoreactive with anti‐type II and type X collagen antibodies. In contrast, at 14 days, TGF‐β1+BMP‐2‐treated aggregates did not contain any morphologically identifiable hypertrophic chondrocytes and their abundant extracellular matrix was not immunoreactive to the anti‐type X collagen antibody. Expression of BMPR‐IA, TGF‐β RI, and TGF‐β RII receptors was detected at all times in each culture condition, indicating that the distinct responses of aggregates to BMP‐2, TGF‐β1 and TGF‐β1+BMP‐2 were not due to overt differences in receptor expression. Collectively, our results suggest that BMP‐2 induces neochondrogenesis of rat periosteum‐derived cells, and that TGF‐β1 modulates the terminal differentiation in BMP‐2 induced chondrogenesis. J. Cell. Biochem. 80:284–294, 2001.

Repair of Osteochondral Defect with Tissue-Engineered Two-Phase Composite Material of Injectable Calcium Phosphate and Hyaluronan Sponge

Articular cartilage has limited capacity for repair. In the present study, tissue-engineered two-phase composite material was used for the repair of osteochondral defects in young adult rabbit knee. This composite material is composed of an injectable calcium phosphate (ICP) and a hyaluronan (HA) derivate of either ACP or HYAFF 11 sponge. The osteochondral defect, 3 mm in diameter and 3 mm deep, was created in the weight-bearing region of the medial femoral condyle. The bone portion of the defect was first filled with ICP to a level approximately 1 mm below the articular surface. HA sponge (3 mm in diameter and 1-1.2 mm thick), with or without loading of autologous bone marrow-derived progenitor cells (MPCs), was then inserted into the defect on top of the ICP as it hardened. Animals were allowed free cage activity postoperatively, and killed 4 or 12 weeks (for the HYAFF 11 sponge group) after the surgery. At 4 weeks, histological examination showed that the defect was filled up to 90-100% of its depth. Whitish repair tissue on the top appeared to be integrated with the surrounding articular cartilage. Four distinct zones of repair tissue were identified: a superficial layer, a chondroid tissue layer, an interface between HA sponge and ICP, and the ICP material. Evidence of extensive osteoclastic and osteoblastic activities was observed in the bone tissue surrounding the defect edge and in ICP material. By 12 weeks, the zonal features of the repair tissue became more distinct; chondrocytes were arranged in a columnar array, and a calcified layer of cartilage was formed beneath the chondroid tissue in some specimens. The healing tissue of the HA sponge material loaded with MPCs had higher cellular density and better integration with the surrounding cartilage than HA sponge material not loaded with MPCs. This study suggests that using a two-phase composite graft may hold potential for the repair of osteochondral defects by providing mechanical support that mimicks subchondral bone, while also providing a chondrogenic scaffold for the top cartilage repair.

Cartilage regeneration using principles of tissue engineering

It is well known that articular cartilage in adults has a limited ability for self-repair. Numerous methods have been devised to augment its natural healing response, but these methods generally lead to filling of the defect with fibrous tissue or fibrocartilage, which lacks the mechanical characteristics of articular cartilage and fails with time. Recently, tissue engineering has emerged as a new discipline that amalgamates aspects from biology, engineering, materials science, and surgery and that has as a goal the fabrication of functional new tissues to replace damaged tissues. The emergence of tissue engineering has facilitated the generation of new concepts and the revival of old ideas all of which has allowed a fresh approach to the repair or regeneration of tissues such as cartilage. The collaborations between scientists with different backgrounds and expertise has allowed the identification of some key principles that serve as the basis for the development of therapeutic approaches that now are less empiric and more hypothesis-driven than ever before. The current authors review some of the considerations regarding the various models used to test and validate the above repair methods and to address different aspects of the cartilage repair paradigm. Also, some key principles identified from past and current research, the need for the development of new biomaterials, and considerations in scale-up of cell-biomaterial constructs are summarized.