Characterization of pro-apoptotic and matrix-degradative gene expression following induction of osteoarthritis in mature and aged rabbits.

OBJECTIVE: The genetic and molecular changes leading to the distinctive alterations of aged cartilage and its propensity for developing osteoarthritis (OA) are unknown. We hypothesized that pro-apoptotic and matrix-degradative gene expression in a rabbit model of induced OA using mature and aged animals might elucidate this relationship. METHODS: Groups of six mature and aged rabbits underwent anterior cruciate ligament transection (ACLT) and were sacrificed 4 weeks after surgery to create an Outerbridge grade II OA. RNA was extracted from the articular cartilage and menisci of the affected knee and was examined with regard to expression of the following genes: Caspase 8, Fas, Fas ligand (Fas-L), p53, aggrecanase, matrix metalloproteinase (MMP)-1, and MMP-3-MMP-13. A second cohort of mature and aged animals was sacrificed with no intervention to the joint and gene expression was assessed in a similar manner. RESULTS: Fas and Caspase 8 showed significantly increased expression in the cartilage of mature animals with induced OA when compared to unoperated controls while induction of OA in aged rabbits did not significantly increase expression of any of the apoptosis genes. Among unoperated animals, the aged cohort showed significantly increased expression of MMP-1 and aggrecanase in cartilage when compared to mature animals. MMP-13 expression was upregulated in aged cartilage following induction of OA. Although ACLT animals showed gross thinning and irregularities within the meniscus, only the expression of Caspase 8 in the aged rabbits was significantly increased after induction of OA. CONCLUSIONS: Aging of articular cartilage shares some qualities with the development of OA, as seen in the parallel increases in gene expression of Caspase 8 and Fas. Although this may imply a common mechanism of cartilage degeneration in aging and OA or even a spectrum of disease, both are complex processes requiring further study.

Kinetics of collagen crosslinking in adult bovine articular cartilage.

OBJECTIVE: Determine the kinetics of collagen crosslinking in adult bovine articular cartilage explants using radiolabel pulse-chase studies. METHODS: Explant cultures of adult bovine articular cartilage were radiolabeled with [14C]lysine in medium including fetal bovine serum and ascorbate, and then maintained for chase periods up to 28 days. In some samples, beta-aminopropionitrile (BAPN) was included during chase to inhibit lysyl oxidase-mediated collagen crosslinking. Tissue was hydrolyzed and analyzed for [14C]metabolites in the forms of lysine, hydroxylysine, dehydrodihydroxylysinonorleucine (DeltaDHLNL), and hydroxylysyl pyridinoline (HP). RESULTS: Explant cultures of adult bovine articular cartilage metabolized lysine into hydroxylysine and the collagen crosslinks, DeltaDHLNL and HP. During chase, [14C]hydroxylysine maintained steady-state levels, [14C]DHLNL rose to a plateau, and [14C]HP increased gradually. Addition of BAPN inhibited formation of [14C]DHLNL. Analysis of raw data and that normalized to [14C]hydroxylysine gave characteristic time constants for formation of DeltaDHLNL and HP crosslinks of 1-2 and 7-30 days, respectively. The distribution of [14C]lysine metabolites in collagen crosslinks was described by peak values in [14C]DHLNL/[14C]hydroxylysine of 0.047-0.064 and in [14C]HP/[14C]hydroxylysine of 0.03. CONCLUSION: Collagen crosslinks form in cartilage explants in vitro according to the classical lysyl oxidase-mediated pathway.

The effect of variations in applied rehabilitation force on collagen concentration and maturation at the intrasynovial flexor tendon repair site.

The biochemical means by which accelerated rehabilitation alters intrasynovial flexor tendon repair site collagen synthesis and extracellular matrix maturation are not fully understood. We hypothesized that an increased level of applied rehabilitative force in a clinically relevant animal model would hasten the maturation of the repair site extracellular matrix as demonstrated by total collagen and collagen cross-link assessment. Twenty-eight flexor digitorum profundus tendons from 14 adult dogs were transected and repaired. The animals received either low- or high-force rehabilitation and were killed 10, 21, and 42 days after surgery. A 10-mm segment of tendon surrounding the repair site was obtained. Biochemical analysis showed that total collagen concentration was significantly reduced at each time point, that the reducible cross-link ratio of dihydroxylysinonorleucine to hydroxylysinonorleucine was significantly increased at each time point, and that the nonreducible pyridinoline cross-link content was significantly decreased at 10 days in both rehabilitative groups. Total collagen content did not vary to a statistically significant degree with either time or as a function of rehabilitation type. Based on these findings several clinically relevant observations can be made. Increasing collagen concentration and repair site maturation do not explain the previously demonstrated increased tensile properties of tendon that occur between 3 and 6 weeks after repair. Higher force rehabilitation does not alter the biochemical composition of the healing tendon through 6 weeks. Coupled with other recent data these findings suggest that high-force rehabilitation does not stimulate accelerated healing after intrasynovial flexor tendon repair.

Chondrocyte apoptosis and regional differential expression of nitric oxide in the medial meniscus following partial meniscectomy.

Partial medial meniscectomy leads to tibial articular cartilage degeneration. Nitric oxide (NO) production increases with the development of osteoarthritis (OA) and has been shown to have a catabolic effect on chondrocytes. Since distribution of chondrocytic and fibroblastic cell types within the total cell population comprising meniscus is region-specific, we compared NO production in the peripheral and central regions of the medial meniscus 12 weeks after partial medial meniscectomy and assessed chondrocyte apoptosis and NO production in the tibial articular cartilage. Additionally, transcriptional gene expression of inducible nitric oxide synthetase (iNOS) and immunohistochemical staining of nitrotyrosine were examined. The results showed that following partial medial meniscectomy, NO production in the central region of the medial meniscus and in the tibial articular cartilage were significantly higher than respective NO levels in normal and sham-operated controls. Reverse transcription polymerase chain reaction (RT-PCR) revealed a high transcriptional expression of the iNOS gene in the central region of the meniscus and in tibial articular cartilage following partial medial meniscectomy. Nitrotyrosine immunoreactivity was prominent in the central region of the medial meniscus and in the deep layer of the tibial articular cartilage and apoptotic cells were also detected in situ in the superficial zone of the tibial articular cartilage and central regions of the medial meniscus following partial medial meniscectomy. These observations suggest that the central region of the meniscus is responsible for NO synthesis associated with apoptosis in both meniscal and articular cartilage cells following partial meniscectomy.

Donor cell fate in tissue engineering for articular cartilage repair.

Articular cartilage repair is a clinical challenge because of its limited intrinsic healing potential. Considerable research has focused on tissue engineering and transplantation of viable chondrogenic cells to enhance cartilage regeneration. However, the question remains: do transplanted allogenic cells survive in the repair with time? This study assessed donor cell fate after transplantation of male New Zealand White rabbit perichondrium cell and polylactic acid constructs into osteochondral defects created in the medial femoral condyles of female New Zealand White rabbits. Repair tissue was harvested at 0, 1, 2, 3, 7, and 28 days after implantation and was evaluated for cell viability and total cell number using confocal microscopic analysis. The number of donor cells in each sample was estimated using quantitative polymerase chain reaction targeting a gender-specific gene present on the Y-chromosome, the sex-determining region Y gene, and a control deoxyribonucleic acid present in male and female cell deoxyribonucleic acid, the matrix metalloproteinase-1 gene promoter. Average cell viability was found to be 87% or more at all times. Donor cells were present in repair tissue for 28 days after implantation. However, the number of donor cells declined from approximately 1 million at Time 0 to approximately 140,000 at 28 days. This decline in donor cells was accompanied by a significant influx of host cells into the repair tissue. This study shows that the sex-determining region Y gene is a valuable marker for tracking the fate of transplanted allogenic cells in tissue engineering.

Biochemical correlates of mTOR inhibition by the rapamycin ester CCI-779 and tumor growth inhibition.

The rapamycin ester, CCI-779, potently inhibits cell growth in vitro, inhibits tumor growth in vivo, and is currently in Phase I clinical trials. To further understand the relationship between plasma systemic exposure and inhibition of the target Ser/Thr kinase, mTOR/FRAP, two assays have been developed. The first assay involves determination of the 4E suppressor protein (4E-BP1) bound to eukaryotic initiation factor 4E (eIF4E), and the second is direct Western analysis of phosphorylation of residue Thr(70) of 4E-BP1. Under normal growth conditions in vitro, rapamycin caused rapid association of 4E-BP1 with eIF4E within 1 h in Rh30 and GC(3) human tumor cells. Association was persistent up to 16 h. In mice, administration of rapamycin (5 or 20 mg/kg) caused rapid association of 4E-BP1 with eIF4E within 4 h in both human colon adenocarcinoma GC(3) and rhabdomyosarcoma Rh30 xenografts. Using phospho-specific antibody against Thr(70) of 4E-BP1, rapid and persistent dephosphorylation within 30 min of exposure to rapamycin was detected in Rh18 rhabdomyosarcoma cells. Evaluation of CCI-779 against Rh18 xenografts showed this tumor to be growth inhibited at daily dose levels of > or =8.7 mg/kg. Because immunoblotting may be more suitable for assaying tumor biopsy tissue, a "blinded" comparison between the effect of CCI-779 on Thr(70) phosphorylation and growth inhibition of human tumor xenografts was undertaken. Mice were treated daily for 5 days with CCI-779 (20 mg/kg/day) or with drug vehicle, and tumor diameters were measured. Tumors were excised 1 h after the final administration and frozen, and phospho Thr(70) was determined by Western blot analysis. The correlation coefficient for decreases in Thr(70) phosphorylation and growth inhibition was high (r(2), 0.99). The results indicate that an assay of decreases in phosphorylation of Thr(70) of 4E-BP1 may be a useful surrogate for determining the inhibition of mTOR activity in tumor specimens.

High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex-vivo gene therapy to repair articular cartilage defects.

BACKGROUND: Primary perichondrial cells and chondrocytes have been used to repair articular cartilage defects in tissue engineering studies involving various animal models. Transfection of these cells with a gene that induces chondrocytic phenotype may form an ideal method to affect tissue engineering of articular cartilage. DESIGN: A protocol for high-efficiency transfection of primary perichondrial and cartilage cells was optimized. Plasmids carrying the marker beta-galactosidase (beta-gal), PTHrP and TGF-beta1 genes driven by a strong mammalian promoter were transfected into primary perichondrial cells and chondrocytes. A three-step method was used to achieve high efficiency of transfection: (1) permeabilization of primary cells using a mild detergent, (2) association of plasmid DNAs with a polycationic (poly-l-lysine) core covalently linked to a receptor ligand (transferrin), (3) introduction of cationic liposomes to form the quaternary complex. For in-vivo assessment, polylactic acid (PLA) scaffolds seeded with beta-gal transfected perichondrial cells were implanted into experimentally created osteochondral defects in rabbit knees for 1 week. RESULTS: The efficiency of transfection was determined to be over 70%in vitro. The transformed cells continued to express beta-gal, in vivo for the entire test period of 7 days. Furthermore, primary perichondrial cells transfected with TGF-beta1 and PTHrP over-expressed their cognate gene products. CONCLUSION: The ability to transfect autologous primary perichondrial cells and chondrocytes with high efficiency using a non-viral system may form a first step towards tissue engineering with these transformed cells to repair articular cartilage defects.

The effects of hyaluronan on the meniscus in the anterior cruciate ligament-deficient knee.

Anterior cruciate ligament (ACL) deficiency often induces meniscal tears and, ultimately, degenerative joint disease. The hypothesis of this study was that hyaluronan (HA; MW = 8 x 105) may have a protective effect on the medial meniscus following a period of ACL deficiency. The animal model consisted of creating an ACL deficiency by ACL transection (ACLT) in 51 mature New Zealand white rabbits. Postoperative injections started 4 weeks after ACLT to allow the ACL deficiency to create a degenerative change in the meniscus. The first group (n = 26) was injected with HA and the second group (n = 25) was injected with vehicle (phosphate-buffered saline) in their ACL-deficient knees once a week for 5 weeks, in a protocol similar to that used clinically. At the end of the injections, the HA-treated menisci showed a reduced meniscus area histomorphometrically (P<0.01), as well as a decrease in water content (P<0.01) when compared with the vehicle-treated menisci. The matrix composition of the menisci was assessed by the total glycosaminoglycans (GAGs) content, which decreased in the vehicle-treated menisci (P<0.05) but did not decrease in the HA-treated menisci. In our model, a positive effect of HA was observed biochemically on the preservation of the meniscus matrix composition in the ACL-deficient knee.

The long-term effects of hyaluronan during development of osteoarthritis following partial meniscectomy in a rabbit model.

OBJECTIVE: The long-term effect of hyaluronan (HA) on meniscus remodeling and articular cartilage preservation was assessed during the development of osteoarthritis following partial meniscectomy in a rabbit model. DESIGN: Approximately 60% of the region of each medial meniscus of 20 rabbit knees was excised bilaterally. The left knee joint was treated with five weekly intraarticular injections of 0.3 ml of HA, beginning 1 week after surgery. The right control knee was injected with PBS on the same schedule. Six months after surgery, animals were killed and the medial menisci and tibial articular cartilage were evaluated morphologically, histologically and biochemically. RESULTS: Meniscal regeneration was observed as newly synthesized translucent tissue, and image analysis revealed that the amount of this tissue was significantly greater in the HA-treated menisci than in the vehicle-treated menisci. Safranin-O staining and image analysis revealed the increased presence of glycosaminoglycans in the HA-treated menisci relative to vehicle-treated menisci while vascularity and biochemical parameters (hydration, total GAGs and reducible collagen crosslinks) were statistically similar in HA- and vehicle-treated menisci. Gross morphologic grading with India ink revealed a trend for less deterioration of tibial articular cartilage in the HA group (P=0.09) while Mankin's score of the HA-treated tibial articular cartilage was marginally lower than that of the vehicle group (P=0.06). Biochemical assessments showed a trend for higher total GAGs concentration in the HA-treated articular cartilage when compared to the vehicle treatment group (P=0.06). CONCLUSION: The present study has demonstrated that following partial meniscectomy, treatment with hyaluronan can enhance meniscal regeneration and may inhibit articular cartilage degeneration as long as six months post surgery.

Transforming growth factor beta one (TGF-beta 1) enhancement of the chondrocytic phenotype in aged perichondrial cells: an in vitro study.

BACKGROUND: Perichondrium is recognized as a tissue with chondrogenic potential yielding cells which can be used for osteochondral repair. Factors which influence the proliferative ability and chondrocytic phenotype of such cells include age and presence of specific growth factors, i.e. TGF-beta 1. The present in vitro study assessed proliferation and markers of chondrocytic phenotype in cells extracted from the rib perichondrium of four- to five-year-old aged rabbits, and assessed the effects of exogenously added TGF-beta 1 on those cells. METHODS: Assays included 3H-thymidine incorporation (cell proliferation), 35S-sulfate incorporation (proteoglycan synthesis) and quantitative RT-PCR for determination of type II collagen gene expression. RESULTS: The results demonstrated that addition of TGF-beta 1 to the culture media stimulated thymidine incorporation and proteoglycan synthesis up to four- and five-fold, respectively, in aged perichondrium-derived cells. Moreover, the exogenous addition of TGF-beta 1 to the culture media resulted in an upregulation of transcriptional expression of the type II collagen gene. CONCLUSIONS: In summary, the present study has demonstrated that exogenously added TGF-beta 1 can stimulate proliferation and chondrocytic phenotype in aged perichondrium-derived cells in vitro.

Fas-dependent and -independent mechanisms of cell death following DNA damage in human colon carcinoma cells.

In thymidylate synthase-deficient (TS-) colon carcinoma cells, thymineless death is mediated via Fas/Fas ligand (FasL) interactions after thymidine deprivation and inhibited by the Fas-inhibitory monoclonal antibody NOK-1. The objective of the study was to elucidate whether other modes of DNA damage induced by doxorubicin, topotecan, and etoposide (VP-16) could elicit a similar cytotoxic response in TS- cells by signaling via the Fas death receptor. After a 72-h drug exposure, a loss in clonogenic survival that was not prevented by NOK-1 was induced by each agent in the absence of acute apoptosis, yielding IC50 values of 5 (doxorubicin), 10 (topotecan), and 150 nM (VP-16). Furthermore, TS- cell clones selected for resistance to Fas-mediated apoptosis (CH-11) were cross-resistant to the induction of thymineless death after thymidine deprivation but were not cross-resistant to doxorubicin, topotecan, or VP-16. A close correlation was found between acute induction of apoptosis (24 h) and up-regulated expression of FasL at high concentrations of each of the three agents (0.3-3 microM doxorubicin, 0.3-3 microM topotecan, and 10-90 microM VP-16), which was caspase dependent but Fas independent. At all drug concentrations, cell cycle distribution analyses demonstrated marked accumulation of cells in the G2-M phase. At nanomolar drug concentrations, prolonged arrest of TS- cells in G2-M phase resulted in the up-regulation of FasL expression and the delayed appearance of apoptotic cells (6 days), which could also be inhibited by the general caspase inhibitor Z-VAD-FMK, but not by NOK-1 or Fas-Fc. In clonogenic assays, Z-VAD-FMK did not rescue cells treated with VP-16 in contrast to treatment with CH-11 or thymineless stress, suggesting an irreversible commitment to cell death in G2-M phase. Expression of FasL at all drug concentrations appeared to be unrelated to the mechanism of drug-induced apoptosis. This was in contrast to the Fas-dependent regulation of thymineless death, which could be inhibited by blocking Fas/FasL interactions.

Regulation of FasL by NF-kappaB and AP-1 in Fas-dependent thymineless death of human colon carcinoma cells.

Cell death due to thymine (dThd) deficiency, associated with the cytotoxic action of 5-fluorouracil in colon cancer, is regulated in thymidylate synthase-deficient (TS(-)) human colon carcinoma cells via the Fas (CD95, APO-1) death receptor. This was demonstrated by inhibiting the loss in clonogenicity of TS(-) cells by anti-FasL and in enhanced survival of TS(-) clones selected for resistance to Fas-mediated apoptosis, following dThd deprivation. During thymineless stress in TS(-) cells, Fas ligand (FasL) is expressed, and its promoter (hFasLPr) is activated. Transactivation of hFasLPr, dependent upon dThd deficiency, was inhibited following mutation of the binding sites for NF-kappaB or AP-1 and by preventing NF-kappaB or AP-1 activation, which inhibited expression of FasL and enhanced clonogenic survival in stable transformants expressing IkappaBalphaM or DN-MEKK, respectively. These results demonstrate the crucial roles for NF-kappaB and AP-1 in the regulation of FasL in Fas-mediated thymineless death of colon carcinoma cells.

The effects of hyaluronan on tissue healing after meniscus injury and repair in a rabbit model.

To assess the effect of hyaluronan on meniscus injury and repair, we had 35 mature New Zealand White rabbits undergo bilateral meniscus injury and repair (19 in the peripheral region, and 16 in the inner region). A longitudinal tear was created in the medial meniscus and repaired with horizontally placed nylon sutures. The left knee joint received intraarticular injections of hyaluronan 1 week after surgery and once a week for 5 weeks. The right knees were injected with phosphate-buffered saline (the carrier vehicle of the hyaluronan). Twelve weeks after repair, tears in the peripheral region showed gross and histologic evidence of healing, with no difference between the vehicle- and hyaluronan-treated menisci. Biochemically, the ratio of reducible collagen cross-links in the hyaluronan-treated menisci was significantly higher than in the vehicle-treated menisci, indicating greater level of collagen remodeling. Biomechanically the vehicle- and hyaluronan-treated menisci demonstrated similarly high tearing load and fracture toughness. In the inner region, poor healing response was observed grossly and histologically in both treatment groups. Water content in the hyaluronan-treated menisci was significantly lower than in the vehicle-treated menisci, indicating a lower level of swelling. Hyaluronan treatment stimulated collagen remodeling in the peripheral region and inhibited swelling of the meniscus repaired in the inner region.

Regulation of alpha(v)beta3 and alpha5beta1 integrin receptors by basic fibroblast growth factor and platelet-derived growth factor-BB in intrasynovial flexor tendon cells.

Integrins are important players in soft tissue healing as molecules that mediate communication between cells and extracellular matrix. Thus, the regulation of the expression of these molecules would be important during wound repair. To explore the regulatory roles of specific growth factors on integrin expression by intrasynovial flexor tendon cells, the present study assessed the in vitro effects of basic fibroblast growth factor and platelet derived growth factor-BB on expression of the alpha5beta1 and alpha(v)beta3 integrins in these cells. Analyses were carried out at the transcriptional (reverse transcription-polymerase chain reaction) and translational (immunohistochemistry) levels of cellular metabolism. Both types of analyses revealed increased expression of alpha5beta1 and alpha(v)beta3 by tendon cells exposed to either basic fibroblast growth factor or platelet-derived growth factor-BB over a wide range of growth factor concentrations employed in the study. Semiquantitative reverse transcription-polymerase chain reaction showed that, relative to control, basic fibroblast growth factor and platelet-derived growth factor-BB increased the expression of alpha(v) mRNA by 2-and 3-fold, respectively. Alpha 5 mRNA expression was also increased 3-fold by basic fibroblast growth factor, and 2-fold by platelet-derived growth factor-BB. We believe the results of this study are significant because the specific integrins affected are intimately involved in two events that have been shown to be important to intrasynovial flexor tendon healing, namely fibronectin deposition (alpha5beta1) as part of the provisional matrix and angiogenesis/revascularization (alpha(v)beta3).

The effects of hyaluronan on matrix metalloproteinase-3 (MMP-3), interleukin-1beta(IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP-1) gene expression during the development of osteoarthritis.

OBJECTIVE: To assess the influence of intra-articular injection of hyaluronan (HA) on expression of matrix metalloproteinase-3 (MMP-3), interleukin-1beta(IL-1beta), and tissue inhibitor of metalloproteinase-1 (TIMP-1) in cartilage and synovium during the process of osteoarthritis (OA). DESIGN: Eighteen mature New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) and were divided into two groups. The first group (HA injection group) received 0.3 ml of intra-articular HA injections into the ACLT knees 4 weeks after transection, once a week for 5 weeks as per clinical treatment presently utilized. The animals in the second group (no injection group) were not injected after ACLT. At death, 9 weeks following surgery, synovium and cartilage were harvested and total RNA was extracted. Gene expressions of MMP-3, IL-1beta and TIMP-1 were analyzed using reverse transcription-polymerase chain reaction (RT-PCR) for each subgroup created according to morphological grade of OA. RESULTS: The extent and grade of cartilage damage in the HA injection group was less severe than in the no injection group. In synovium, expression of MMP-3 and IL-1beta mRNA was suppressed in the mild grades of OA in the HA injection group. HA treatment had either no effect on MMP-3 expression in cartilage at all grades of OA or on enhanced MMP-3 and IL-1beta expression in synovium at a progressed grade. No effect of HA treatment on TIMP-1 expression was observed in either cartilage or synovium. CONCLUSIONS: These results suggest that one of the mechanism of therapeutic effect of HA is down-regulation of MMP-3 and IL-1beta in synovium during early development of OA.

Integrative cartilage repair: inhibition by beta-aminopropionitrile.

The effects of beta-aminopropionitrile, a known inhibitor of lysyl oxidase, on the extractability of newly synthesized collagen and integrative cartilage repair were determined in explant cultures of adult bovine articular cartilage. Dose-escalation studies indicated that treatment of cartilage explants for 6 days with beta-aminopropionitrile caused a dose-dependent inhibition of proteoglycan synthesis ([35S]sulfate incorporation) with a 50% inhibition at 2.2 mM. However, 0.25 mM beta-aminopropionitrile had no detectable effect on proteoglycan synthesis and was thus used for subsequent experiments. Treatment of cartilage with beta-aminopropionitrile for 14 days increased the extractability of newly synthesized collagen with 4 M guanidine-HCl while having little effect on proteoglycan synthesis, proteoglycan deposition, collagen synthesis (formation of [3H]hydroxyproline after labeling with [3H]proline), collagen deposition, or cartilage cellularity (DNA content). In untreated cultures, the percentage of radiolabeled collagen ([3H]hydroxyproline) that was extractable after 1 day of radiolabeling, 6 days of radiolabeling, or 6 days of label and 6 days of chase decreased from 81 to 25 and 9%, respectively. In beta-aminopropionitrile-treated cultures, the extractability was relatively higher (96, 62, and 47%, respectively). Treatment with beta-aminopropionitrile after radiolabeling with [14C]lysine also significantly inhibited the formation of the reducible crosslink [14C]dihydroxylysinonorleucine without affecting the overall deposition in cartilage of [14C]lysine and [14C]hydroxylysine. In functional repair studies, treatment with beta-aminopropionitrile caused an almost complete inhibition of integration between pairs of cartilage explants maintained in apposition for 2 weeks. These results indicate that beta-aminopropionitrile blocks the formation of collagen crosslinks in cartilage explants and suggest that such crosslinks are critical to integrative cartilage repair.

Inhibition of apoptosis after thymineless stress is conferred by oncogenic K-Ras in colon carcinoma cells.

Ras functions as a molecular switch for several downstream targets and may promote either apoptosis or survival dependent upon the cell system and stimulus. The functional significance of a transfected K-Ras oncogene in influencing apoptosis induced by thymineless stress was examined in a thymidylate synthase (TS)-deficient (TS-) colon carcinoma cell line derived from GC3/c1 after thymidine deprivation. Oncogenic K-Ras conferred survival in TS- K-Ras clones compared with TS- (untransfected) and TS- pCIneo (vector control). Previously, we had demonstrated that thymineless death involved signaling via Fas/FasL interactions. However, in the presence of oncogenic K-Ras, survival did not involve down-regulation of Fas or FasL expression but did involve members of the Bcl-2 family. Bcl-2 and Bax expression remained relatively constant during thymineless stress in all cell lines. Apoptosis in the presence of wild-type Ras correlated with up-regulated expression of Bak that did not occur in TS- K-Ras clones, whereas survival in these clones correlated with elevated expression of Bcl-xL. Thus, the Bak:Bcl-xL ratio was high in TS- and TS- pCIneo cells undergoing apoptosis, whereas the Bcl-xL:Bak ratio was high in TS- K-Ras clones exhibiting a survival response.

Growth factor expression in healing rabbit medial collateral and anterior cruciate ligaments.

Exogenously administered growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF-beta) and basic fibroblast growth factor (bFGF) have been shown to affect connective tissue healing in vivo, but their intrinsic role in the healing response has not been established. In the present study, immunohistochemistry with antibodies directed against these growth factors showed that expression of PDGF, TGF-beta 1 and bFGF was increased in and around the wound site in the rabbit medial collateral ligament (MCL) seven days following surgical injury. The strong expression of PDGF correlated with the observed increased cellularity consistent with this growth factor's mitogenic and chemotactic properties. Expression of these growth factors was also increased in wounded rabbit anterior cruciate ligaments (ACL) at seven days following surgical injury, but such expression was limited to the edge of the ACL injury site and was of lesser intensity relative to the MCL. This study suggests that PDGF and TGF-beta 1, and to a lesser extent bFGF, are actively involved during the early stage of MCL healing, but have a more limited presence in the injured rabbit ACL.

Long-term effects of hyaluronan on experimental osteoarthritis in the rabbit knee.

OBJECTIVE: Long-term assessment of the effect of hyaluronan (HA) on the articular cartilage and synovium in an animal knee joint during the development of osteoarthritis (OA). DESIGN: Sixty mature New Zealand white rabbits underwent unilateral anterior cruciate ligament transection (ACLT) and were divided into two groups. Group 1 (SA) received intra-articular injections of 0.3 ml hyaluronan (HA) (i.e., ARTZ, MW: 8 x 10(5)) beginning 4 weeks after ACLT, once a week for 5 weeks. Group 2 (SV) received injections of the vehicle (phosphate buffered saline) in the same fashion as with the SA group. The contralateral nonoperated knee served as control. All animals were killed 21 weeks after surgery and their knee joints evaluated by gross morphologic, histologic, histomorphometric and biochemical analyses. RESULTS: Gross morphological inspection indicated that the femoral condyles from the knees injected with vehicle suffered more severe cartilage damage than cartilage from the knees injected with HA. Furthermore, two out of three histomorphometric parameters measured in the HA-treated cartilage (i.e., cartilage thickness and cartilage area which were not statistically different than control) provided evidence showing a protective effect of HA on the femoral condyles following ACLT. Biochemical analysis showed articular cartilage remaining on the femoral condyles following ACLT to have similar characteristics to contralateral controls. However, DNA concentration in the synovium from the ACLT knees of the vehicle-treated animals was greater than contralateral control, while this parameter was not statistically different than contralateral control in the HA treated animals. CONCLUSIONS: These results demonstrate a protective effect of HA on preservation of the articulating surface of the femoral condyle following ACLT up to 21 weeks post-surgery.

Architectural remodeling in deep frozen meniscal allografts after total meniscectomy.

This study assesses the biological characteristics of cell repopulation and matrix remodeling in deep frozen meniscal allografts after total meniscectomy and allograft transplantation. Thirty-two mature New Zealand White rabbits were operated on, and the allografts were assessed at 12 and 26 weeks postsurgery. Before transplantation, fresh medial menisci were placed into liquid nitrogen to kill all intrinsic cells. Frozen meniscal allografts showed collagen remodeling coincident with revascularization and cellular repopulation. Biochemical characterization showed active collagen remodeling of the allografts at 12 and 26 weeks. In situ hybridization showed that expression of types I and III procollagen mRNAs were increased at 12 weeks, especially near the region of the synovial capsule. At 26 weeks after transplantation, the pattern of collagen-type expression progressed toward normal; that is, type I procollagen mRNA expression was prominent, whereas only a small amount of type III procollagen mRNA was observed. These data indicate the adaptation of the repopulating cells from the host to the frozen allograft at 26 weeks after transplantation.