Effect of short-term enzymatic treatment on cell migration and cartilage regeneration: in vitro organ culture of bovine articular cartilage.

Depending on the damage extent and adjacent tissue condition in traumatic cartilage injury, it is possible to heal the tissue by resident cells. Unlike autologous chondrocyte implantation, short-term enzymatic treatment is an effective single-step procedure without extra cell expansion. Moreover, this method has been shown to significantly increase cellularity in lesion edges, resulting in enhanced integration and interfacial strength. We hypothesize that the locally digested extracellular matrix by treatment allows effortless cell migration from the adjacent tissue. Full-thickness cartilage discs and osteochondral explants were prepared from mature bovine stifle joints. These specimens were treated with collagenase in a culture medium. Two concentrations, 0.25 and 0.5 mg/mL, were used with various treating time of 10, 30, and 180 min. The cartilages were subsequently washed and cultured with fibrin hydrogel. The effect of enzymatic treatment on cell migration was apparent in both experiments of the cartilage disc and full-thickness cartilage defect model. In the disc culture, the treatment resulted in an approximately three to four times higher number of migrated cells than nontreated control. In short-term collagenase-treated groups, the proteoglycan (PG) loss was localized in the edge of tissue with minimal cell death. The treatment also accelerated cell migration in the full-thickness cartilage defects and some cells differentiated into chondrocytes with the deposit of PG. Gene expression results could support the characteristics of migrated cells, which had migratory ability and chondrogenic differentiation potential with overexpression of collagen type I and II, respectively. Based on these results, short-term enzymatic treatment, which can accelerate cell migration into traumatically injured cartilage, has great potential for clinical application.

Biocompatibility and preclinical feasibility tests of a temperature-sensitive hydrogel for the purpose of surgical wound pain control and cartilage repair.

We recently introduced a novel pluronic F127 and hyaluronic acid-based hydrogel (HG) designed to deliver a broad range of therapeutics. The reverse-thermal responsive HG exhibits physical properties that seem to be ideal for the local delivery of drug- and cell-based therapies to specific anatomic sites through percutaneous injection. However, questions related to the HG's safety and efficacy must first be addressed. To address these issues, we performed standard in vitro cytotoxicity and drug release tests and in vivo biocompatibility tests in a rat model. In addition, we determined whether the HG was an effective stem cell carrier in a rat cartilage defect model. We found that the HG showed viability and biocompatibility levels similar to those reported for F127 or hyaluronic acid alone. In vitro drug release studies with bupivacaine, a drug used clinically for local pain relief, revealed that after an initial burst bupivacaine was released continuously for 10 days. Stem cells loaded in the HG were retained in situ and stimulated cartilage regeneration in experimental defects. Taken as a whole, these findings support further efforts to develop the HG as a versatile system for the delivery of a wide range of therapeutic agents in humans. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

Organ culture stability of the intervertebral disc: rat versus rabbit.

There is a need to develop mechanically active culture systems to better understand the role of mechanical stresses in intervertebral disc (IVD) degeneration. Motion segment cultures that preserve the native IVD structure and adjacent vertebral bodies are preferred as model systems, but rapid ex vivo tissue degeneration limits their usefulness. The stability of rat and rabbit IVDs is of particular interest, as their small size makes them otherwise suitable for motion segment culture. The goal of this study was to determine if there are substantial differences in the susceptibility of rat and rabbit IVDs to culture-induced degeneration. Lumbar IVD motion segments were harvested from young adult male Sprague-Dawley rats and New Zealand White rabbits and cultured under standard conditions for 14 days. Biochemical assays and safranin-O histology showed that while glycosaminoglycan (GAG) loss was minimal in rabbit IVDs, it was progressive and severe in rat IVDs. In the rat IVD, GAG loss was concomitant with the loss of notochordal cells and the migration of endplate (EP) cells into the nucleus pulposus (NP). None of these changes were evident in the rabbit IVDs. Compared to rabbit IVDs, rat IVDs also showed increased matrix metalloproteinase-3 (MMP-3) and sharply decreased collagen type I and II collagen expression. Together these data indicated that the rabbit IVD was dramatically more stable than the rat IVD, which showed culture-related degenerative changes. Based on these findings we conclude that the rabbit motion segments are a superior model for mechanobiologic studies.

Biocompatibility and preclinical feasibility tests of a temperature-sensitive hydrogel for the purpose of surgical wound pain control and cartilage repair.

We recently introduced a novel pluronic F127 and hyaluronic acid-based hydrogel (HG) designed to deliver a broad range of therapeutics. The reverse-thermal responsive HG exhibits physical properties that seem to be ideal for the local delivery of drug- and cell-based therapies to specific anatomic sites through percutaneous injection. However, questions related to the HG's safety and efficacy must first be addressed. To address these issues, we performed standard in vitro cytotoxicity and drug release tests and in vivo biocompatibility tests in a rat model. In addition, we determined whether the HG was an effective stem cell carrier in a rat cartilage defect model. We found that the HG showed viability and biocompatibility levels similar to those reported for F127 or hyaluronic acid alone. In vitro drug release studies with bupivacaine, a drug used clinically for local pain relief, revealed that after an initial burst bupivacaine was released continuously for 10 days. Stem cells loaded in the HG were retained in situ and stimulated cartilage regeneration in experimental defects. Taken as a whole, these findings support further efforts to develop the HG as a versatile system for the delivery of a wide range of therapeutic agents in humans.

Chondrogenic progenitor cells respond to cartilage injury.

OBJECTIVE: Hypocellularity resulting from chondrocyte death in the aftermath of mechanical injury is thought to contribute to posttraumatic osteoarthritis. However, we observed that nonviable areas in cartilage injured by blunt impact were repopulated within 7-14 days by cells that appeared to migrate from the surrounding matrix. The aim of this study was to assess our hypothesis that the migrating cell population included chondrogenic progenitor cells that were drawn to injured cartilage by alarmins. METHODS: Osteochondral explants obtained from mature cattle were injured by blunt impact or scratching, resulting in localized chondrocyte death. Injured sites were serially imaged by confocal microscopy, and migrating cells were evaluated for chondrogenic progenitor characteristics. Chemotaxis assays were used to measure the responses to chemokines, injury-conditioned medium, dead cell debris, and high mobility group box chromosomal protein 1 (HMGB-1). RESULTS: Migrating cells were highly clonogenic and multipotent and expressed markers associated with chondrogenic progenitor cells. Compared with chondrocytes, these cells overexpressed genes involved in proliferation and migration and underexpressed cartilage matrix genes. They were more active than chondrocytes in chemotaxis assays and responded to cell lysates, conditioned medium, and HMGB-1. Glycyrrhizin, a chelator of HMGB-1 and a blocking antibody to receptor for advanced glycation end products (RAGE), inhibited responses to cell debris and conditioned medium and reduced the numbers of migrating cells on injured explants. CONCLUSION: Injuries that caused chondrocyte death stimulated the emergence and homing of chondrogenic progenitor cells, in part via HMGB-1 release and RAGE-mediated chemotaxis. Their repopulation of the matrix could promote the repair of chondral damage that might otherwise contribute to progressive cartilage loss.

Selection of reference genes for normalization of quantitative real-time PCR in organ culture of the rat and rabbit intervertebral disc.

BACKGROUND: The accuracy of quantitative real-time RT-PCR (qRT-PCR) is often influenced by experimental artifacts, resulting in erroneous expression profiles of target genes. The practice of employing normalization using a reference gene significantly improves reliability and its applicability to molecular biology. However, selection of an ideal reference gene(s) is of critical importance to discern meaningful results. The aim of this study was to evaluate the stability of seven potential reference genes (Actb, GAPDH, 18S rRNA, CycA, Hprt1, Ywhaz, and Pgk1) and identify most stable gene(s) for application in tissue culture research using the rat and rabbit intervertebral disc (IVD). FINDINGS: In vitro, four genes (Hprt1, CycA, GAPDH, and 18S rRNA) in rat IVD tissue and five genes (CycA, Hprt1, Actb, Pgk1, and Ywhaz) in rabbit IVD tissue were determined as most stable for up to 14 days in culture. Pair-wise variation analysis indicated that combination of Hprt1 and CycA in rat and the combination of Hprt1, CycA, and Actb in rabbit may most stable reference gene candidates for IVD tissue culture. CONCLUSIONS: Our results indicate that Hprt1 and CycA are the most stable reference gene candidates for rat and rabbit IVD culture studies. In rabbit IVD, Actb could be an additional gene employed in conjunction with Hprt1 and CycA. Selection of optimal reference gene candidate(s) should be a pertinent exercise before employment of PCR outcome measures for biomedical research.