Gregory G. Reinholz

Animal models for cartilage reconstruction.

Animal models are widely used to develop and evaluate tissue-engineering techniques for the reconstruction of damaged human articular cartilage. For the purpose of this review, these model systems will include in vitro culture of animal cells and explants, heterotopic models of chondrogenesis, and articular cartilage defect models. The objectives for these preclinical studies are to engineer articular cartilage for the functional restoration of a joint surface that appears anatomically, histologically, biologically, biochemically, and mechanically to resemble the original joint surface. While no animal model permits direct application to humans, each is capable of yielding principles on which decisions can be made that might eventually translate into a human application. Clearly, the use of animal models has and will continue to play a significant role in the advancement of this field. Each animal model has specific advantages and disadvantages. The key issue in the selection of an appropriate animal model is to match the model to the question being investigated and the hypothesis to be tested. The purpose of this review is to discuss issues regarding animal model selection, the benefits and limitations of these model systems, scaffold selection with emphasis on polymers, and evaluation of the tissue-engineered articular cartilage.

Distinct mechanisms of bisphosphonate action between osteoblasts and breast cancer cells: identity of a potent new bisphosphonate analogue

While the effects of bisphosphonates on bone-resorbing osteoclasts have been well documented, the effects of bisphosphonates on other cell types are not as well studied. Recently, we reported that bisphosphonates have direct effects on bone-forming human fetal osteoblast cells (hFOB) [1]. In this report, the role of the mevalonate pathway in the actions of bisphosphonates on hFOB, and MDA-MB-231 human breast cancer cells was examined. These studies included a novel bisphosphonate analog, the anhydride formed between arabinocytidine 5′ phosphate and etidronate (Ara-CBP). Ara-CBP was the most potent inhibitor of hFOB and MDA-MB-231 cell proliferation, and stimulator of hFOB cell mineralization compared to etidronate, the anhydride formed between AMP and etidronate (ABP), pamidronate, and zoledronate. Inhibition of hFOB cell proliferation by Ara-CBP and zoledronate was partially reversed by mevalonate pathway intermediates, and stimulation of hFOB cell mineralization was completely reversed by mevalonate pathway intermediates. These results suggest that zoledronate and Ara-CBP act, at least in part, via inhibition of the mevalonate pathway in hFOB cells. In contrast, none of the mevalonate pathway intermediates reversed the inhibition of MDA-MB-231 cell proliferation by the bisphosphonates, or the effects of pamidronate on hFOB cells. As a positive control, the effects of mevastatin on hFOB and MDA-MB-231 cells were completely reversed by mevalonate. In summary, these data suggest that zoledronate and Ara-CBP induce human osteoblast differentiation via inhibition of the mevalonate pathway. In contrast, the inhibition of MDA-MB-231 cell proliferation by the bisphosphonates appears to be through mechanisms other than inhibition of the mevalonate pathway.

Tissue engineering of cartilage using poly-ɛ-caprolactone nanofiber scaffolds seeded in vivo with periosteal cells

OBJECTIVE To determine the potential of periosteal cells to infiltrate poly-epsilon-caprolactone (PCL) nanofiber scaffolds in vivo and subsequently produce cartilage in vitro. DESIGN PCL nanofiber scaffolds, with or without chitosan-coating were implanted under periosteum in 6-month-old rabbits. Transforming growth factor-beta1 (TGF-beta1) or vehicle was injected into each implant site. After 1, 3, 5 or 7 days, scaffolds were removed, separated from the periosteum, and the scaffolds and periosteum were cultured separately for 6 weeks under chondrogenic conditions. Sulfated glycosaminoglycan (GAG), type II collagen, DNA content, cartilage yield, and calcium deposition were then analyzed. RESULTS Cell infiltration was observed in all scaffolds. Cartilage formation in the uncoated scaffolds increased with duration of implantation (maximum at 7 days). Cells in the uncoated scaffolds implanted for 7 days produced significantly higher levels of both GAG [560 (95% confidence interval (CI), 107-1013) vs 228 (95% CI, 177-278) microg GAG/microg DNA] and cartilage yield [9% (95% CI, 3-14%) vs 0.02% (95% CI, 0-0.22%)] compared to chitosan-coated scaffolds (P=0.006 or less). There was no significant difference in GAG content or cartilage yield between the TGF-beta1-injected and vehicle-injected scaffolds. However, significantly more mineral deposition was detected in TGF-beta1-injected scaffolds compared to vehicle-injected scaffolds (P<0.0001). Cartilage yield from the periosteum, moreover, was significantly increased by subperiosteal TGF-beta1 injections (P<0.001). However, this response was reduced when chitosan-coated scaffolds were implanted. CONCLUSIONS This study demonstrates that it is possible to seed PCL nanofiber scaffolds with periosteal cells in vivo and subsequently produce engineered cartilage in vitro.

The effect of scaffold composition on the early structural characteristics of chondrocytes and expression of adhesion molecules

Previously we demonstrated that chondrocyte ECM synthesis and mitotic activity was dependent on scaffold composition when cultured on uncoated PCL scaffolds (pPCL) or PCL composites containing hyaluronan (PCL/HA), chitosan (PCL/CS), fibrin (PCL/F), or collagen type I (PCL/COL1). We hypothesized that initial cell contact with these biomaterials results in ultrastructural changes and alters CD44 and integrin beta1 expression. The current study was designed to investigate the early ultrastructural responses of chondrocytes on these scaffolds and expression of CD44 and integrin beta1. A common observation 1 h after seeding was the abundance of cell processes. Different types of cell processes occurred in different areas of the same cell and on different cells within the same composite. Chondrocytes seeded onto PCL/CS had the greatest cell surface enhancement. PCL/HA promoted CD44 expression and almost spherical cells with a low degree of surface enhancement. PCL/COL1 enabled continuing expression of integrin beta1 and CD44. In contrast, cells in PCL/CS, PCL/F and pPCL promoted elliptic cells with a higher degree of surface enhancement and no prolonged CD44 and integrin beta1 expression. A strong variability of cell surface processes indicated either reparative or degenerative adaptation to the artificial environment. Interestingly, we found initial integrin beta1 expression in all composite scaffolds, but not in pPCL although this promoted strong adhesiveness as indicated by the formation of stress fibers. In conclusion, chondrocytes respond to biomaterials early after implantation by altering ultrastructural characteristics and expression of CD44 and integrin beta1.

Porous tantalum and poly-ε-caprolactone biocomposites for osteochondral defect repair: Preliminary studies in rabbits

Currently, various techniques are in use for the repair of osteochondral defects, none of them being truly satisfactory and they are often two step procedures. Comorbidity due to cancellous bone harvest from the iliac crest further complicates the procedure. Our previous in vitro studies suggest that porous tantalum (TM) or poly‐ε‐caprolactone scaffolds (PCL) in combination with periosteal grafts could be used for osteochondral defect repair. In this in vivo study, cylindrical osteochondral defects were created on the medial and lateral condyles of 10 rabbits and filled with TM/periosteum or PCL/periosteum biosynthetic composites (n = 8 each). The regenerated osteochondral tissue was then analyzed histologically, and evaluated in an independent and blinded manner by five different observers using a 30‐point histological score. The overall histological score for PCL/periosteum was significantly better than for TM/periosteum. However, most of the regenerates were well integrated with the surrounding bone (PCL/periosteum, n = 6.4; TM/periosteum, n = 7) along with partial restoration of the tidemark (PCL/periosteum, n = 4.4; TM/periosteum, n = 5.6). A cover of hyaline‐like morphology was found after PCL/periosteum treatment (n = 4.8), yet the cartilage yields were inconsistent. In conclusion, the applied TM and PCL scaffolds promoted excellent subchondral bone regeneration. Neo‐cartilage formation from periosteum supported by a scaffold was inconsistent. This is the first study to show in vivo results of both PCL and TM scaffolds for a novel approach to osteochondral defect repair.

Poly‐ϵ‐caprolactone/gel hybrid scaffolds for cartilage tissue engineering

The aim of this study was to determine the suitability of hybrid scaffolds composed of naturally derived biopolymer gels and macroporous poly-epsilon-caprolactone (PCL) scaffolds for neocartilage formation in vitro. Rabbit articular chondrocytes were seeded into PCL/HA (1 wt % hyaluronan), PCL/CS (0.5 wt % chitosan), PCL/F (1:3 fibrin sealant plus aprotinin), and PCL/COL1 (0.24% type I collagen) hybrids and cultured statically for up to 50 days. Growth characteristics were evaluated by histological analysis, scanning electron microscopy, and confocal laser scanning microscopy. Neocartilage was quantified using a dimethyl-methylene blue assay for sulfated glycosaminoglycans (sGAG) and an enzyme-linked immunosorbent assay for type II collagen (COL2), normalized to dsDNA content by fluorescent PicoGreen assay. Chondrocytes were homogenously distributed throughout the entire scaffold and exhibited a predominantly spheroidal shape 1 h after being seeded into scaffolds. Immunofluorescence depicted expanding proteoglycan deposition with time. The sGAG per dsDNA increased in all hybrids between days 25 and 50. PCL/HA scaffolds consistently promoted highest yields. In contrast, total sGAG and total COL2 decreased in all hybrids except PCL/CS, which favored increasing values and a significantly higher total COL2 at day 50. Overall, dsDNA content decreased significantly with time, and particularly between days 3 and 6. The PCL/HA hybrid displayed two proliferation peaks at days 3 and 25, and PCL/COL1 displayed one proliferation peak at day 12. The developed hybrids provided distinct short-term environments for implanted chondrocytes, with not all of them being explicitly beneficial (PCL/F, PCL/COL1). The PCL/HA and PCL/CS hybrids, however, promoted specific neocartilage formation and initial cell retention and are thus promising for cartilage tissue engineering.

Rejuvenation of periosteal chondrogenesis using local growth factor injection

OBJECTIVE To examine the potential for rejuvenation of aged periosteum by local injection of transforming growth factor-beta1 (TGF-beta1) and insulin-like growth factor-1 (IGF-1) alone or in combination to induce cambium cell proliferation and enhance in vitro periosteal cartilage formation. METHODS A total of 367 New Zealand white rabbits (6, 12, and 24+ month-old) received subperiosteal injections of TGF-beta1 and/or IGF-1 percutaneously. After 1, 3, 5, or 7 days, the rabbits were sacrificed and cambium cellularity or in vitro cartilage forming capacity was determined. RESULTS A significant increase in cambium cellularity and thickness, and in vitro cartilage formation was observed after injection of TGF-beta1 alone or in combination with IGF-1. In 12 month-old rabbits, mean cambium cellularity increased 5-fold from 49 to 237 cells/mm and in vitro cartilage production increased 12-fold from 0.8 to 9.7 mg 7 days after TGF-beta1 (200 ng) injection compared to vehicle controls (P<0.0001). A correlation was observed between cambium cellularity and in vitro cartilage production (R2=0.98). An added benefit of IGF-1 plus TGF-beta1 on in vitro cartilage production compared to TGF-beta1 alone was observed in the 2 year-old rabbits. IGF-1 alone generally had no effect on either cambium cellularity or in vitro cartilage production in any of the age groups. CONCLUSIONS These results clearly demonstrate that it is possible to increase cambium cellularity and in vitro cartilage production in aged rabbit periosteum, to levels comparable to younger rabbits, using local injection of TGF-beta1 alone or in combination with IGF-1, thereby rejuvenating aged periosteum.

Chondrogenic differentiation of bone marrow‐derived mesenchymal stromal cells via biomimetic and bioactive poly‐ε‐caprolactone scaffolds

The objective of this study was to develop a scaffold for mesenchymal stromal cell (MSC) recruitment, proliferation, and chondrogenic differentiation. The concept behind the design is to mimic the cartilage matrix and contain stimulatory agents that make continuous supply of inductive factors redundant. Nanofibrous (N: ~400 nm) and microfibrous (M: ~10 μm) poly-ε-caprolactone (PCL) scaffolds were combined with 1% high-molecular-weight sodium hyaluronate (NHA/MHA), 1% hyaluronan (HA) and 200 ng transforming growth factor-beta 1 (TGF-β1; NTGF/MTGF), or 0.1% bovine serum albumin (N/M). Scaffolds were seeded with MSCs from bone marrow and cultured without growth factors in vitro. Cultures with chondrogenic medium supplemented with TGF-β1 served as controls. Proliferation, migration, and release of TGF-β1 were investigated. Cell differentiation was evaluated by polymerase chain reaction (PCR) and real-time PCR. NTGF and MTGF exhibited primarily an initial release of TGF-β1. None of the factors released by the scaffolds recruited MSCs. The expression of aggrecan was dependent on the scaffold ultrastructure with nanofibers promoting increasing and microfibers decreasing expression levels. Composites containing HA demonstrated elevated seeding efficiency and lower type I collagen expression. Expression of type II collagen was dependent on continuous or late supply of TGF-β1, which was not provided by our scaffold design. The initial release of TGF-β1 induced an expression of type I collagen and osteogenic marker genes. In conclusion, nanofibrous PCL scaffolds with or without augmentation are suitable for chondrogenic initiation of MSCs. Initial release of HA is sufficient in terms of directing the implanted MSCs toward a chondrogenic end, whereas a late release of TGF-β1 is preferred to foster type II and avoid type I collagen expression.

Directional fluid flow enhances in vitro periosteal tissue growth and chondrogenesis on poly-ε-caprolactone scaffolds†

The purpose of this study was to investigate the effect of directional fluid flow on periosteal chondrogenesis. Periosteal explants were harvested from 2-month-old rabbits and sutured onto poly-epsilon-caprolactone (PCL) scaffolds with the cambium layer facing away from the scaffolds. The periosteum/PCL composites were cultured in suspension in spinner flask bioreactors and exposed to various fluid flow velocities: 0, 20, 60, and 150 rpm for 4 h each day for 6 weeks. The application of fluid flow significantly increased percent cartilage yield in periosteal explants from 17% in the static controls to 65-75% under fluid flow (there was no significant difference between 20, 60, or 150 rpm). The size of the neocartilage was also significantly greater in explants exposed to fluid flow compared with static culture. The development of zonal organization within the engineered cartilage was observed predominantly in the tissue exposed to flow conditions. The Youngs modulus of the engineered cartilage exposed to 60 rpm was significantly greater than the samples exposed to 150 and 20 rpm. These results demonstrate that application of directional fluid flow to periosteal explants secured onto PCL scaffolds enhances cell proliferation, chondrogenic differentiation, and cell organization and alters the biomechanical properties of the engineered cartilage.

Transforming growth factor-β1 modulates insulin-like growth factor binding protein-4 expression and proteolysis in cultured periosteal explants

OBJECTIVE Periosteum is involved in bone growth and fracture healing and has been used as a cell source and tissue graft for tissue engineering and orthopedic reconstruction including joint resurfacing. Periosteum can be induced by transforming growth factor beta (TGF-beta) or insulin-like growth factor-I (IGF-I) alone or in combination to form cartilage. However, little is known about the interaction between IGF and TGF-beta signaling during periosteal chondrogenesis. The purpose of this study was to determine the effect of TGF-beta1 on IGF binding protein-4 (IGFBP-4) and the IGFBP-4 protease pregnancy-associated plasma protein-A (PAPP-A) expression in cultured periosteal explants. DESIGN Periosteal explants from rabbits were cultured with or without TGF-beta1. IGFBP-4 and PAPP-A mRNA levels were determined by real-time quantitative PCR. Conditioned medium was analyzed for IGFBP-4 and PAPP-A protein levels and IGFBP-4 protease activity. RESULTS TGF-beta1-treated explants contained lower IGFBP-4 mRNA levels throughout the culture period with a maximum reduction of 70% on day 5 of culture. Lower levels of IGFBP-4 protein were also detected in the conditioned medium from TGF-beta1-treated explants. PAPP-A mRNA levels were increased 1.6-fold, PAPP-A protein levels were increased threefold, and IGFBP-4 protease activity was increased 8.5-fold between 7 and 10days of culture (the onset of cartilage formation in this model) in conditioned medium from TGF-beta1-treated explants. CONCLUSIONS This study demonstrates that TGF-beta1 modulates the expression of IGFBP-4 and PAPP-A in cultured periosteal explants.