Indomethacin induces differential effects on in vitro endochondral ossification depending on the chondrocyte's differentiation stage

Heterotopic ossification (HO) is the abnormal formation of bone in soft tissues and is a frequent complication of hip replacement surgery. Heterotopic ossifications are described to develop via endochondral ossification and standard treatment is administration of indomethacin. It is currently unknown how indomethacin influences heterotopic ossification on a molecular level; therefore, we aimed to determine whether indomethacin might influence heterotopic ossification via impairing the chondrogenic phase of endochondral ossification. Progenitor cell models differentiating in the chondrogenic lineage (ATDC5, primary human bone marrow stem cells and ex vivo periosteal agarose cultures) were treated with increasing concentrations of indomethacin and a decrease in gene‐ and protein expression of chondrogenic and hypertrophic markers (measured by RT‐qPCR and immunoblotting) as well as decreased glycosamino‐glycan content (by alcian blue histochemistry) was observed. Even when hypertrophic differentiation was provoked, the addition of indomethacin resulted in decreased hypertrophic marker expression. Interestingly, when mature chondrocytes were treated with indomethacin, a clear increase in collagen type 2 expression was observed. Similarly, when ATDC5 cells and bone marrow stem cells were pre‐differentiated to obtain a chondrocyte phenotype and indomethacin was added from this time point onward, low concentrations of indomethacin also resulted in increased chondrogenic differentiation. Indomethacin induces differential effects on in vitro endochondral ossification, depending on the chondrocyte's differentiation stage, with complete inhibition of chondrogenic differentiation as the most pronounced action. This observation may provide a rational behind the elusive mode of action of indomethacin in the treatment of heterotopic ossifications. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:847–857, 2017.

     

Comparative immunolocalisation of perlecan, heparan sulphate, fibroblast growth factor-18, and fibroblast growth factor receptor-3 and their prospective roles in chondrogenic and osteogenic development of the human foetal spine

Purpose

A comparative immunolocalisation study of perlecan, HS, FGF-18 and FGFR-3 in the 12–20-week gestational age human foetal spine was undertaken to identify spatiotemporal associations between these components to provide insights into prospective roles in spinal development.

Methods

Comparative immunolocalisations of matrix and cell associated components in Histochoice-fixed paraffin-embedded human foetal spinal tissues.

Results

The 12–14-week-old human foetal spine was a predominantly cartilaginous structure with the discs displaying a relative paucity of proteoglycan compared to the adjacent cartilaginous vertebral rudiments, notochordal remnants were also observed. HS and perlecan had a widespread distribution throughout the spine at 12 weeks, however, FGF-18 was only localised to the outer AF margins and hypertrophic cell condensations in the vertebral bodies. This contrasted with HS distributions at 14–20 weeks, which were prominent in the developing intervertebral disc (IVD). Ossification centres were also evident centrally within the vertebral rudiments surrounded by small columns of hypertrophic chondrocytes which expressed FGFR-3 and FGF-18 and upregulated levels of perlecan. FGF-18 also had a prominent localisation pattern in the developing IVD and the cartilaginous endplate while FGFR-3 was expressed throughout the disc interspace. This suggested roles for perlecan, FGF-18 and FGFR-3 in chondrogenic and osteogenic events which drive discal development and ossification of the vertebral bodies.

Conclusions

The above data supported a role for FGF-18 in discal development and in the terminal osteogenic differentiation of chondroprogenitor cell populations, which promote vertebral ossification during spinal development.     

Deletion of estrogen receptor beta accelerates early stage of bone healing in a mouse osteotomy model

Summary

This study examined the role of estrogen receptor (ER) beta during mouse femoral fracture healing by employing ER knockout (KO) mice. The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification.

Introduction

This study was conducted to examine the role of ER beta during fracture healing.

Methods

Female ERbeta knockout (KO) mice (18 weeks old) and age-matched female wild-type (WT) mice underwent open osteotomy on the right femur. They were sacrificed at 1, 2, 4 and 6 weeks post-fracture. The sera and callus samples were subjected to the following analyses: micro-computed tomography (CT)-based angiography, micro-CT evaluation, histological examination, histomorphometry examination, real-time polymerase chain reaction (PCR) analysis, biochemical marker, and mechanical testing.

Results

Micro-CT-based angiography showed that the total vessel volume at the fracture site was larger in the KO group than the WT group at 1 and 2 weeks post-fracture. Micro-CT analysis revealed that the callus volume was significantly higher in the KO group from week 2 to week 4 post-fracture when compared with the WT group consistent with the histological data. Analysis of biochemical markers indicated that circulating P1NP levels in the KO mice were significantly higher than in the WT mice from week 2 to week 4 and that temporal expression of circulating C-terminal telopeptide of type I collagen (CTX) levels was also higher in the KO mice than in the WT mice. These results were consistent with quantitative real-time PCR analysis. The ultimate load, stiffness, and energy to failure were significantly higher in the KO mice than in the WT mice at week 4.

Conclusions

The fracture healing in KO mice was enhanced in the early stage of neovascularization and the middle stage of endochondral ossification, but not by the end of healing. Blockade of ERbeta can be considered as another therapeutic strategy for osteoporotic fracture and non-union fracture.     

Effect of parathyroid hormone-related protein in an in vitro hypertrophy model for mesenchymal stem cell chondrogenesis

Purpose

Mesenchymal stem cells (MSCs) express markers of hypertrophic chondrocytes during chondrogenic differentiation. We tested the suitability of parathyroid hormone-related protein (PTHrP), a regulator of chondrocyte hypertrophy in embryonic cartilage development, for the suppression of hypertrophy in an in vitro hypertrophy model of chondrifying MSCs.

Methods

Chondrogenesis was induced in human MSCs in pellet culture for two weeks and for an additional two weeks cultures were either maintained in standard chondrogenic medium or transferred to a hypertrophy-enhancing medium. PTHrP(1–40) was added to the medium throughout the culture period at concentrations from 1 to 1,000 pM. Pellets were harvested on days one, 14 and 28 for biochemical and histological analysis.

Results

Hypertrophic medium clearly enhanced the hypertrophic phenotype, with increased cell size, and strong alkaline phosphatase (ALP) and type X collagen staining. In chondrogenic medium, 1–100 pM PTHrP(1–40) did not inhibit chondrogenic differentiation, whereas 1,000 pM PTHrP(1–40) significantly reduced chondrogenesis. ALP activity was dose-dependently reduced by PTHrP(1–40) at 10–1,000 pM in chondrogenic conditions. Under hypertrophy-enhancing conditions, PTHrP(1–40) did not inhibit the induction of the hypertrophy. At the highest concentration (1,000 pM) in the hypertrophic group, aggregates were partially dedifferentiated and differentiated areas of these aggregates maintained their hypertrophic appearance.

Conclusions

PTHrP(1–40) treatment dose-dependently reduced ALP expression in MSC pellets cultured under standard chondrogenic conditions and is thus beneficial for the maintenance of the chondrogenic phenotype in this medium condition. When cultured under hypertrophy-enhancing conditions, PTHrP(1–40) could not diminish the induced enhancement of hypertrophy in the MSC pellets.     

Heterotopic Bone Formation About the Hip Undergoes Endochondral Ossification: A Rabbit Model

Background

Heterotopic ossification (HO) occurs most commonly after trauma and surgery about the hip and may compromise subsequent function. Currently available animal models describing the cellular progression of HO are based on exogenous osteogenic induction agents and may not reflect the processes following trauma.

Questions/purposes

We therefore sought to characterize the histologic progression of heterotopic bone formation in an animal model that recapitulates the human condition without the addition of exogenous osteogenic material.

Methods

We used a rabbit model that included intramedullary instrumentation of the upper femur and ischemic crush injury of the gluteal muscle. Bilateral surgical induction procedures were performed on 30 animals with the intention of inciting the process of HO; no supplemental osteogenic stimulants were used. Three animals were sacrificed at each of 10 predetermined times between 1 day and 26 weeks postoperatively and the progression of tissue maturation was graded histologically using a five-item scale.

Results

Heterotopic bone reliably formed de novo and consistently followed a pathway of endochondral ossification. Chondroid elements were found in juxtaposition with immature woven bone in all sections that contained mature osseous elements.

Conclusions

These results establish that HO occurs in an animal model mimicking the human condition following surgical trauma about the hip; it is predictable in its histologic progression and follows a pathway of endochondral bone formation.

Clinical Relevance

By showing a consistent pathway of endochondral ossification leading to ectopic bone formation, this study provides a basis for understanding the mechanisms by which HO might be mitigated by interventions.     

Anti-osteoporosis therapy and fracture healing

Background

A number of medications are approved for treatment of osteoporosis. As mode of action usually is anti-catabolic/anti-resorptive or anabolic, it is of interest to know whether these drugs affect not only normal bone remodeling, but also fracture healing.

Objective

The purpose of this paper is to give a short overview of the potential effect of various anti-osteoporotic medication on fracture healing.

Methods

A narrative literature review was performed to describe the current knowledge.

Results

Anti-catabolic/anti-resorptive drugs: for bisphosphonates, the most common class of drugs in this group, experimental studies have shown a larger and stronger callus and delayed remodeling but no evidence of delayed healing. A human monoclonal antibody to RANKL is another anti-catabolic drug, with the only report to date showing enhanced healing in an animal model. Strontium ranelate is a drug where both anti-catabolic and a weak anabolic effect have been proposed, with experimental data ranging from no effect to significant increase in both callus volume and strength. Anabolic drugs: PTH has demonstrated accelerated healing of various experimental fractures and of distal radius and pelvic fractures in humans. While the exact mechanism is not fully understood, PTH results in increased recruitment and differentiation of chondrocytes and enhancement of endochondral ossification. A monoclonal antibody to block sclerostin is another potential anabolic pathway, where animal data have shown increase in bone mass and strength. The potential effect on fracture healing is yet to be studied.

Conclusion

There are still large gaps in the understanding of the potential effect of anti-osteoporotic drugs on fracture healing, although based on present knowledge a recent or present fracture should not be considered as a contraindication to such treatment.     

Comparison between normal and loose fragment chondrocytes in proliferation and redifferentiation potential

Purpose

Loose fragments in osteochondritis dissecans (OCD) of the knee require internal fixation. On the other hand, loose fragments derived from spontaneous osteonecrosis of the knee (SONK) are usually removed. However, the difference in healing potential between OCD- and SONK-related loose fragments has not been elucidated. In this study, we investigated proliferative activity and redifferentiation potential of normal cartilage-derived and loose fragment-derived chondrocytes.

Methods

Cells were prepared from normal articular cartilages and loose fragment cartilages derived from knee OCD and SONK. Cellular proliferation was compared. Redifferentiation ability of pellet-cultured chondrocytes was assessed by real-time PCR analyses. Mesenchymal differentiation potential was investigated by histological analyses. Positive ratio of a stem cell marker CD166 was evaluated in each cartilaginous tissue.

Results

Normal and OCD chondrocytes showed a higher proliferative activity than SONK chondrocytes. Chondrogenic pellets derived from normal and OCD chondrocytes produced a larger amount of safranin O-stained proteoglycans compared with SONK-derived pellets. Expression of chondrogenic marker genes was inferior in SONK pellets. The CD166-positive ratio was higher in normal cartilages and OCD loose fragments than in SONK loose fragments.

Conclusions

The OCD chondrocytes maintained higher proliferative activity and redifferentiation potential compared with SONK chondrocytes. Our results suggest that chondrogenic properties of loose fragment-derived cells and the amount of CD166-positive cells may affect the repair process of osteochondral defects.     

Expression of Indian Hedgehog During Fracture Healing in Adult Rat Femora

Indian hedgehog (Ihh) has recently been shown to be expressed in prehypertrophic and hypertrophic chondrocytes during embryonic development, and it has been implicated in the regulation of terminal differentiation of chondrocytes. In this paper we examined the expression of Ihh during fracture healing in an adult rat model. A transverse diaphyseal fracture was made in the right femur, and the expression of Ihh in the fracture callus was examined at 1, 2, and 3 weeks after fracture. Northern blot analysis demonstrated the expression of Ihh mRNA in these tissues. Immunohistological analysis detected hedgehog protein in prehypertrophic chondrocytes in the fracture callus at 1 week after fracture. From 2 weeks and on, positive staining was observed in hypertrophic chondrocytes as well. At 3 weeks, some of the osteoblasts close to the endochondral ossification front were also stained positive for hedgehog protein. Our data indicate that Ihh is expressed in chondrocytes and osteoblasts during the process of fracture healing in adult rat femora, suggesting that Ihh, a regulator of endochondral ossification in embryonic development, may also play a role in the regulation of bone formation during fracture repair in adult animals. Received: 29 March 1999 / Accepted: 30 September 1999     

Programmed removal of chondrocytes during endochondral fracture healing

This investigation tested the hypothesis that the removal of chondrocytes during endochondral fracture healing involves an ordered process of programmed cell death. To accomplish this, unilateral closed fractures were created in the femora of 36 Sprague-Dawley rats. The rats were killed in groups of four on days 1, 3, 7, 14, 21, 28, 42, 49, and 56 after fracture. The femora were embedded in paraffin and tested for expression of specific markers of fragmented DNA with use of a terminal deoxyuridyl transferase-mediated deoxyuridine triphosphatebiotin nick end labeling (TUNEL) technique. To determine the potential for trans–differentiation of chondrocytes to osteoblasts calluses were also hybridized to detect expression of osteocal in mRNA. Cell proliferation was assessed by an immunohistochemical detection method for proliferating cell nuclear antigen. A separate group of four rats was killed on day 28 to represent the later stage of the endochondral ossification, and the calluses were examined for cellular morphology with transmission electron microscopy. The results showed a coordination in both time and space of the activities of cellular proliferation and programmed cell death. Cell proliferation was most active in the earlier phases of fracture healing (days 1 through 14) although TUNEL expression was apparent in hypertrophic chondrocytes on day 14 after fracture and persisted until day 28. In the later stages of fracture healing (days 14 through 28), proliferating cell nuclear antigen was no longer synthesized in hard callus (intramembranous bone) and cell removal was the dominant activity in soft callus chondrocytes. Expression of osteocalcin mRNA was detected in osteoblasts but not in hypertrophic chondrocytes or in any other nonosteoblastic cell type. These findings support the hypothesis that the removal of chondrocytes during endochondral fracture healing is part of an ordered transition of tissue types in which the cellular mechanisms are genetically programmed to involve proliferation, maturation, and apoptotic cell death.     

The experimental research of transforming growth factor-beta 1 expression during fracture healing]

OBJECTIVE: To observe the effect of TGF-beta 1 in the regulation of fracture healing. METHOD: The expression of transforming growth factor-beta 1 (TGF-beta 1) in different period of fracture healing was investigated by immunohistochemistry. RESULTS: It was found that the expression of TGF-beta 1 changed in different period. The cells in the cambial layer of the periostlum showed low or negative signal in the immediate injury response period. The osteoblasts differentiated from the periosteum cells stained strongly in the intramembranous ossification period, and the differentiated chondrocytes stained most strongly in the chondrogenesis period. The hypertrophic chondrocytes showed negative signal and the osteoblasts stained strongly in the endochondral ossification period. These results suggested that the expression of TGF-beta 1 was closely related to the proliferation and differentiation state of repair cells. CONCLUSION: TGF-beta 1 is intimately involved in the control of fracture healing.     

Cellular reactions to biodegradable magnesium alloys on human growth plate chondrocytes and osteoblasts

Purpose

In recent decades operative fracture treatment using elastic stable intramedullary nails (ESINs) has mainly taken precedence over conservative alternatives in children. The development of biodegradable materials that could be used for ESINs would be a further step towards treatment improvement. Due to its mechanical and elastic properties, magnesium seems to be an ideal material for biodegradable implant application. The aim of this study was therefore to investigate the cellular reaction to biodegradable magnesium implants in vitro.

Methods

Primary human growth plate chondrocytes and MG63 osteoblasts were used for this study. Viability and metabolic activity in response to the eluate of a rapidly and a slower degrading magnesium alloy were investigated. Furthermore, changes in gene expression were assessed and live cell imaging was performed.

Results

A superior performance of the slower degrading WZ21 alloy’s eluate was detected regarding cell viability and metabolic activity, cell proliferation and morphology. However, the ZX50 alloy’s eluate induced a favourable up-regulation of osteogenic markers in MG63 osteoblasts.

Conclusions

This study showed that magnesium alloys for use in biodegradable implant application are well tolerated in both osteoblasts and growth plate chondrocytes respectively.     

Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis

Background

Microfracture is a first-line treatment option for cartilage repair. In microfracture, subchondral mesenchymal cortico-spongious progenitor cells (CSP) enter the defect and form cartilage repair tissue. The aim of our study was to investigate the effects of joint disease conditions on the in vitro chondrogenesis of human CSP.

Methods

CSP were harvested from the subchondral bone marrow. CSP characterization was performed by analysis of cell surface antigen pattern and by assessing the chondrogenic, osteogenic and adipogenic differentiation potential, histologically. To assess the effect of synovial fluid (SF) on chondrogenesis of CSP, micro-masses were stimulated with SF from healthy (ND), osteoarthritis (OA) and rheumatoid arthritis donors (RA) without transforming growth factor beta 3.

Results

CSP showed the typical cell surface antigen pattern known from mesenchymal stem cells and were capable of osteogenic, adipogenic and chondrogenic differentiation. In micro-masses stimulated with SF, histological staining as well as gene expression analysis of typical chondrogenic marker genes showed that SF from ND and OA induced the chondrogenic marker genes aggrecan, types II and IX collagen, cartilage oligomeric matrix protein (COMP) and link protein, compared to controls not treated with SF. In contrast, the supplementation with SF from RA donors decreased the expression of aggrecan, type II collagen, COMP and link protein, compared to CSP treated with SF from ND or OA.

Conclusion

These results suggest that in RA, SF may impair cartilage repair by subchondral mesenchymal progenitor cells in microfracture, while in OA, SF may has no negative, but a delaying effect on the cartilage matrix formation.     

Expression of parathyroid hormone-related peptide and insulin-like growth factor I during rat fracture healing.

Parathyroid hormone-related peptide (PTHrP) and insulin-like growth factor I (IGF-I) are both involved in the regulation of bone and cartilage metabolisms and their interaction has been reported in osteoblasts. To investigate the interaction of PTHrP and IGF-I during fracture healing, the expression of mRNA for PTHrP and IGF-I, and receptors for PTH/PTHrP and IGF were examined during rat femoral fracture healing using an in situ hybridization method and an immunohistochemistry method, respectively. During intramembranous ossification, PTHrP mRNA, IGF-I mRNA and IGF receptors were detected in preosteoblasts, differentiated osteoblasts and osteocytes in the newly formed trabecular bone. PTH/PTHrP receptors were markedly detected in osteoblasts and osteocytes, but only barely so in preosteoblasts. During cartilaginous callus formation, PTHrP mRNA was expressed by mesenchymal cells and proliferating chondrocytes. PTH/PTHrP receptors were detected in proliferating chondrocytes and early hypertrophic chondrocytes. IGF-I mRNA and IGF receptor were co-expressed by mesenchymal cells, proliferating chondrocytes, and early hypertrophic chondrocytes. At the endochondral ossification front, osteoblasts were positive for PTHrP and IGF-I mRNA as well as their receptors. These results suggest that IGF-I is involved in cell proliferation or differentiation in mesenchymal cells, periosteal cells, osteoblasts and chondrocytes in an autocrine and/or paracrine fashion. Furthermore, PTHrP may be involved in primary callus formation presumably co-operating with IGF-I in osteoblasts and osteocytes, and by regulating chondrocyte differentiation in endochondral ossification.     

Structured three-dimensional co-culture of mesenchymal stem cells with chondrocytes promotes chondrogenic differentiation without hypertrophy

Objective

This study investigated a novel approach to induce chondrogenic differentiation of human mesenchymal stem cells (hMSC). We hypothesized that a structured three-dimensional co-culture using hMSC and chondrocytes would provide chondroinductive cues to hMSC without inducing hypertrophy.

Method

In an effort to promote optimal chondrogenic differentiation of hMSC, we created bilaminar cell pellets (BCPs), which consist of a spherical population of hMSC encased within a layer of juvenile chondrocytes (JC). In addition to histologic analyses, we examined proteoglycan content and expression of chondrogenic and hypertrophic genes in BCPs, JC pellets, and hMSC pellets grown in the presence or absence of transforming growth factor-β (TGFβ) following 21 days of culture in either growth or chondrogenic media.

Results

In either growth or chondrogenic media, we observed that BCPs and JC pellets produced more proteoglycan than hMSC pellets treated with TGFβ. BCPs and JC pellets also exhibited higher expression of the chondrogenic genes Sox9, aggrecan, and collagen 2A1, and lower expression of the hypertrophic genes matrix metalloproteinase-13, Runx2, collagen 1A1, and collagen 10A1 than hMSC pellets. Histologic analyses suggest that JC promote chondrogenic differentiation of cells in BCPs without hypertrophy. Furthermore, when cultured in hypoxic and inflammatory conditions intended to mimic the injured joint microenvironment, BCPs produced significantly more proteoglycan than either JC pellets or hMSC pellets.

Conclusion

The BCP co-culture promotes a chondrogenic phenotype without hypertrophy and, relative to pellet cultures of hMSCs or JCs alone, is more resistant to the adverse conditions anticipated at the site of articular cartilage repair.     

Immunohistochemical detection of activin A,follistatin, and activin receptors during fracture healing in the rat

Activins are multifunctional proteins that belong to the transforming growth factor-β superfamily and are thought to play an important role in modulating the formation of bone. Activins exert their cellular effects by way of activin type-I and type-II serine/threonine kinase receptors. Follistatin is an activin-binding protein that can suppress the biological effects of activins. In this study, the immunohistochemical expression of activin A, follistatin, and activin receptors was studied during fracture healing in the rat. Activin A was weakly detected in the periosteum near the fracture ends at an early stage but was absent in the chondrocytes around the fracture gap, where endochondral ossification took place. An antibody to follistatin stained osteogenic cells in the periosteum near the fracture ends; moderate and strong staining were observed in proliferating, mature, and hypertrophied chondrocytes at the sites of endochondral ossification. Levels of activin A and follistatin were high near the posteoblasts on the surface of the newly formed trabecular bone. In addition, an intense localization of activin A was noted where multinucleated osteoclast-like cells were present. This study suggests that the activin-follistatin system may contribute to cellular events related to the formation and remodeling of bone during fracture healing. Activin type-I and type-II receptors were co-expressed in intramembranous and endochondral ossification sites. The expression of activin type-I, type-II, and type-IIB receptors in the absence of activin A in the endochondral ossification suggests that other isoforms of activins may signal by way of these receptors.     

Temporal and spatial expression of bone morphogenetic proteins in extracorporeal shock wave-promoted healing of segmental defect

Extracorporeal shock wave (ESW) is a noninvasive acoustic wave, which has recently been demonstrated to promote bone repair. The actual healing mechanism triggered by ESW has not yet been identified. Bone morphogenetic proteins (BMP) have been implicated as playing an important role in bone development and fracture healing. In this study, we aimed to examine the involvement of BMP-2, BMP-3, BMP-4, and BMP-7 expression in ESW promotion of fracture healing. Rats with a 5-mm segmental femoral defect were given ESW treatment using 500 impulses at 0.16 mJ/mm(2). Femurs and calluses were subjected to immunohistochemistry and RT-PCR assay 1, 2, 4, and 8 weeks after treatment. Histological observation demonstrated that fractured femurs received ESW treatment underwent intensive mesenchymal cell aggregation, hypertrophic chondrogenesis, and endochondral/intramembrane ossification, resulting in the healing of segmental defect. Aggregated mesenchymal cells at the defect, chondrocytes at the hypertrophic cartilage, and osteoblasts adjunct to newly formed woven bone showed intensive proliferating cell nuclear antigen expression. ESW treatment significantly promoted BMP-2, BMP-3, BMP-4, and BMP-7 mRNA expression of callus as determined by RT-PCR, and BMP immunoreactivity appeared throughout the bone regeneration period. Mesenchymal cells and immature chondrocytes showed intensive BMP-2, BMP-3, and BMP-4 immunoreactivity. BMP-7 expression was evident on osteoblasts located at endochondral ossification junction. Our findings suggest that BMP play an important role in signaling ESW-activated cell proliferation and bone regeneration of segmental defect.     

Expression of p21CIP1/WAF1 in Chondrocytes

During endochondral ossification, proliferative activity of chondrocytes is arrested and the cells undergo terminal hypertrophic differentiation. We examined the expression of the cyclin-dependent kinase inhibitor, p21^CIP1/WAF1 in permanent cartilage (xyphoid and articular cartilage) and in cartilage undergoing endochondral ossification (growth plate, epiphyseal ossification centers, and costochondral junctions) to determine if p21 is up-regulated in chondrocytes during hypertrophic differentiation. Northern blot analyses demonstrated expression of p21 in chondrocytes undergoing endochondral ossification and from sites of permanent cartilage. Quantitative analyses of Northern data showed an association between expression of the hypertrophic-specific marker, collagen type X, and the level of 21 expression. In situ hybridization of rodent femoropatellar joints and costochondral junctions localized p21 mRNA to chondrocytes within both the proliferative and hypertrophic zones of the growth plates, in chondrocytes involved in formation of the epiphyseal ossification centers, and in articular chondrocytes. Immunohistochemical analyses of p21 expression in the same tissues demonstrated comparatively higher levels of p21 protein in postmitotic chondrocytes. These data suggest that p21 is active in cell cycle regulation in chondrocytes, and that increased p21 expression is associated with hypertrophic differentiation. Received: 11 October 1996 / Accepted: 23 April 1997     

Distinct and Overlapping Patterns of Localization of Bone Morphogenetic Protein (BMP) Family Members and a BMP Type II Receptor During Fracture Healing in Rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.     

An ovine in vitro model for chondrocyte-based scaffold-assisted cartilage grafts

Background

Scaffold-assisted autologous chondrocyte implantation is an effective clinical procedure for cartilage repair. From the regulatory point of view, the ovine model is one of the suggested large animal models for pre-clinical studies. The aim of our study was to evaluate the in vitro re-differentiation capacity of expanded ovine chondrocytes in biomechanically characterized polyglycolic acid (PGA)/fibrin biomaterials for scaffold-assisted cartilage repair.

Methods

Ovine chondrocytes harvested from adult articular cartilage were expanded in monolayer and re-assembled three-dimensionally in PGA-fibrin scaffolds. De- and re-differentiation of ovine chondrocytes in PGA-fibrin scaffolds was assessed by histological and immuno-histochemical staining as well as by real-time gene expression analysis of typical cartilage marker molecules and the matrix-remodelling enzymes matrix metalloproteinases (MMP) -1, -2 and ?13 as well as their inhibitors. PGA scaffolds characteristics including degradation and stiffness were analysed by electron microscopy and biomechanical testing.

Results

Histological, immuno-histochemical and gene expression analysis showed that dedifferentiated chondrocytes re-differentiate in PGA-fibrin scaffolds and form a cartilaginous matrix. Re-differentiation was accompanied by the induction of type II collagen and aggrecan, while MMP expression decreased in prolonged tissue culture. Electron microscopy and biomechanical tests revealed that the non-woven PGA scaffold shows a textile structure with high tensile strength of 3.6 N/mm² and a stiffness of up to 0.44 N/mm², when combined with gel-like fibrin.

Conclusion

These data suggest that PGA-fibrin is suited as a mechanically stable support structure for scaffold-assisted chondrocyte grafts, initiating chondrogenic re-differentiation of expanded chondrocytes.