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Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2

Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair –... Background: Articular cartilage has little capacity for repair in vivo, however, a small number of studies have shown that, in vitro, a damage/repair response can be induced. Recent work by our group has shown that cartilage can respond to single impact load and culture by producing repair cells on the articular surface. The purpose of this study was to identify whether chondrocyte outgrowth into a 3D scaffold could be observed following single impact load and culture. The effect of bone morphogenic-2 (BMP-2) on this process was investigated. Methods: Cartilage explants were single impact loaded, placed within a scaffold and cultured for up to 20 days +/- BMP-2. Cell numbers in the scaffold, on and extruding from the articular surface were quantified and the immunohistochemistry used to identify the cellular phenotype. Results: Following single impact load and culture, chondrocytes were observed in a 3D gelatin scaffold under all culture conditions. Chondrocytes were also observed on the articular surface of the cartilage and extruding out of the parent cartilage and on to the cartilage surface. BMP-2 was demonstrated to quantitatively inhibit these events. Conclusion: These studies demonstrate that articular chondrocytes can be stimulated to migrate out of parent cartilage following single impact load and culture. The addition of BMP-2 to the culture medium quantitatively reduced the repair response. It may be that the inhibitory effect of BMP-2 in this experimental model provides a clue to the apparent inability of articular cartilage to heal itself following damage in vivo. Cartilage repair is an important clinical challenge and is Background Articular cartilage constantly experiences the damaging being studied extensively. effects of biomechanical 'wear and tear' and is reported to have little capacity for permanent repair due primarily to Work undertaken by our group has recently described a its limited proliferative capability [1]. This limited repair novel intrinsic damage repair response in mature equine capacity leads to damage accumulation, resulting in the articular cartilage explants following single impact load loss of cartilage integrity and the development of chronic and subsequent culture [3]. This response is characterised degenerative joint disease, specifically osteoarthritis (OA) by the appearance of chondrocyte repair cells on damaged [2]. cartilage. This apparent mobilisation of chondrocytes has Page 1 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 also been described following significant cartilage damage Impact loading of cartilage discs (mincing), where the chondrocytes were observed to be Discs were randomly divided into two groups – control migrating into a 3D scaffold. This scaffold was subse- (unimpacted) or impacted. Discs were impacted using a quently used to repair cartilage lesions in vivo [4]. This drop tower device following the method described previ- work demonstrates that cartilage can be stimulated suffi- ously [3,11,12]. Each disc, with the articular surface facing ciently to mobilise chondrocytes to sites of damage and down was impacted from a height of 2.5 cm using a beyond, into a scaffold, and that these mobilised cells weight of 500 g. The approximate impact energy applied have distinct and exciting clinical possibilities. to each disc was 0.175 J, impacted at a velocity of approx- imately 0.7 m/s. To ensure constant compression condi- In the damage/repair model described by our group [3] tions, the impactor was left upon the disc for 10 s before we have shown that repair cell numbers are significantly being removed. increased when the cartilage is cultured in the presence of 50 ng/ml fibroblastic growth factor-2 (FGF-2). The mech- In vitro culture of cartilage discs anism by which repair cells appear on the surface of dam- After impact, discs were placed into pockets created by aged cartilage is unknown, however we have previously sharp excision in a gelatin scaffold (Gelfoam, Pharmacia hypothesised that FGF-2 (both exogenous and from Upjohn, USA) (1 cm × 1 cm pieces). Cartilage+Gelfoam endogenous release [5,6]) is driving this process. FGF-2 is units were cultured for 0, 10 and 20 days in Dulbecco's one of a family of growth factors that have profound modified Eagle's medium (DMEM, Sigma-Aldrich, UK) effects on cartilage development and repair. Other growth supplemented with 200 IU/ml penicillin (Invitrogen, factors are also clearly involved in cartilage biology and of UK), 2.5 υg/ml streptomycin (Invitrogen, UK), 500 υg/ml these the bone morphogenic proteins (BMPs) are some of ascorbic acid (Sigma Aldrich, UK) and 10% fetal calf the most widely studied. In particular it has been shown serum (Invitrogen, UK) at 37 degrees C and 5% CO2.) that BMP-2 promotes chondrogenesis [7], induces the dif- Groups of discs were also cultured in the presence of 100 ferentiation of stem cells into chondrocytes [8] is shown ng/ml BMP-2 (Sigma Aldrich, UK). 100 ng/ml BMP-2 was to be up-regulated in cartilage after mechanical damage used in this experiment as 100 ng/ml has been shown to [9] and promotes articular cartilage repair experimentally be an effective dose for stimulation of chondrocyte and [10]. BMP-2 is therefore a growth factor of potential inter- mesenchymal stem cell responses [13,14]. Each experi- est in in vitro cartilage repair. mental time point was made up of 3 cartilage discs i.e each experiment was performed in triplicate in each of the The aims of this study were (i) to identify whether repair three animals. Control explant discs (not impacted) were cells produced following SIL and subsequent culture have cultured for the same time-periods. At the end of each the ability to migrate out of parent cartilage into a 3D gel- time point cartilage discs were embedded in Tissue Tek atin scaffold and ii) to investigate the effects of 100 ng/ml OCT (Sakura Finetek Europe, The Netherlands) and snap- BMP-2 on these events. frozen in liquid nitrogen or placed immediately into for- mal saline and paraffin embedded. Frozen sections (10 μm) were cut and placed on poly-L-lysine coated slides for Methods Horses histological and immunocytochemical analysis. Cartilage was obtained from horses aged between 7 and 9 years (n = 3) that were humanely destroyed for reasons Histological and immunocytochemical analysis other than joint disease. For this study, only normal, Sections were stained for routine histological analysis healthy cartilage was used based on the lack of pathology with Haematoxylin and Eosin (Sigma Aldrich). The fol- following macroscopical and microscopical examination. lowing features of each experimental time point were quantified cells with elongated shape, cells with pyknotic Harvesting of cartilage nuclei, cells on the surface of the section, cells extruded Cartilage discs (7 mm diam.) were dissected aseptically from the section and cells trapped in gelfoam. The quan- from the articular surface of the proximal phalanx, with- tification of each of these cell features was performed by out attached subchondral bone, using a sterile cork borer. counting 3 adjacent high power fields in the top, middle Discs were placed into sterile phosphate-buffered saline and bottom of the cartilage section using a standardised (PBS) containing antibiotics and antimycotics (200 IU/ml grid approach. penicillin, 2.5 μg/ml fungizone, 100 μg/ml streptomycin, 20 μg/ml gentamycin, Invitrogen, Paisley, Scotland), and Identification of the synthesis of hyaline cartilage specific then washed a further 3 times in sterile PBS. proteins was performed by standard immunolocalisation using the following primary antibodies – polyclonal rab- bit anti-rat collagen IX/XI (Calbiochem/Merck), polyclo- nal rabbit anti-porcine collagen type II (raised and Page 2 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 characterised by M. E. Davies), polyclonal mouse anti- bovine collagen type I(Sigma-Aldrich) and polyclonal goat anti-rabbit fibronectin (Sigma, UK). These antibod- ies have been previously shown to recognise equine epitopes ([15,16]). The appropriate FITC-conjugated sec- ondary antibodies were used to visualise the antigen. Quantitation techniques Data from all sections was pooled. Chondrocyte numbers were expressed as the percentage of cells as a total of the number of cells within the section. This calculated figure was used in order to minimise the effect of different cells numbers within sections from different animals. Statisti- cal significance was identified suing Mann-Whitney and Kruskal-Wallis tests. Results Cartilage damage following a single impact load His la Figure 1 r su tolrfa ogical s ce and the gelfoam sc ection showing th affold e junction between the articu- Cartilage discs that had been impacted suffered damage at Histological section showing the junction between the articu- the articular surface immediately upon impact. This dam- lar surface and the gelfoam scaffold. Cells have migrated out age was characterised by loss of proteoglycan (as demon- of the cartilage (bottom left of picture) and are clearly seen strated by loss of metachromatic staining), roughening of associated with the gelatin 'fibres' The arrow marks the Gel- the articular surface and fissure formation, as previously foam-cartilage junction. Stained with H&E. ×200. described ([17,18]). All control explants, in contrast, pre- sented an intact articular surface and uniform metachro- matic staining, with no apparent loss of proteoglycan. Quantification of chondrocytes in scaffold At t = 0 all chondrocytes were observed to be within the parent bone as this time point represents the intial state of the cartilage prior to culture. When cultured, chondro- cytes were observed within the gelfoam scaffold at day 11 and day 20 under all experimental conditions, including non impacted cartilage. Chondrocytes were usually observed to be closely associated with gelfoam 'fibres' sug- gesting that they may be adherent to the scaffold (Figure 1). The chondrocytes observed within the gelfoam at d11 and d 20 were quantified as a percentage of cells within the parent section. There was no significant difference between the numbers of chondrocytes in the gelfoam between control and SIL sections at d11 or d20, however there was a statistically significant difference decrease in the samples cultured with BMP-2 (p = 0.01 at day 11 com- Grap fo Figure 2 ld (h to show n 'Gelfoam') umbers of cells captured in 3D gelatin scaf- pared to SIL sections, p = 0.049 compared to control sec- Graph to show numbers of cells captured in 3D gelatin scaf- tions, p = 0.03 at day 20 compared to SIL sections, p = fold ('Gelfoam'). It can be seen that cells were detected in the 0.03 compared to control sections). When the number of Gelfoam scaffold at days 11 and 20 in all three culture condi- chondrocytes within the gelfoam at days 11 and 20 was tions. At day 11 and day 20 there were significantly reduced compared, it was shown that the percentage of chondro- numbers of chondrocytes in the Gelfoam in the samples cul- cytes in the gelfoam had significantly decreased between tured in the presence of 100 ng/ml BMP-2 (*). At day 20 from d11 to d20 in control (P = 0.005) and SIL (P = there was a significant decrease in cell numbers in all three 0.007) sections (Figure 2). There was no significant experimental conditions. change in the number of cells that had entered the gel- Page 3 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 foam scaffold from the sections supplemented with BMP- 2 over time. Cells extruded from sections Cells were considered to be extruded from the parent car- tilage if they could be observed actually exiting out of the articular surface (Figure 3). In all cases there appeared to be a breach in the articular surface through which cyto- plasm was apparently moving. Chondrocytes were observed to be extruding from the parent cartilage at days 11 and 20 under all culture conditions studied (Figure 4). At t = 0 no chondrocytes were observed to be extruding i.e. extrusion of cells from the cartilage did not appear to occur prior to culturing. There was no significant differ- ence in the number of cells extruding from the parent car- tilage in control and SIL cartilage, however, in the SIL+BMP sections there was a statistically significant decrease in the number of cells extruding at day 11 com- Grap la Figure 4 r car h to show n tilage surface umbers of cells extruding out of the articu- pared to controls (p = 0.04) and SIL sections (p = 0.05) Graph to show numbers of cells extruding out of the articu- (Figure 4). lar cartilage surface. It can be seen that cells were observed to be extruding at days 11 and 20 in all three culture condi- Cells on the articular surface tions. At day 11 there was a significant decrease in the No cells were detected on the articular surface at day 0. number of cells extruding from the cartilage surface in the Cells were detected on the articular surface at day 11 and samples cultured in the presence of 100 ng/ml BMP-2 (*) day 20 under all experimental conditions (Figure 5). compared to both control and SIL sections. There was no significant difference in the number of cells on the articular surface between control and SIL cartilage at either day 11 or 20, however the number of cells on the articular surface of the SIL+BMP-2 sections were statisti- cally decreased compared to both control and SIL, (p = 0.03 at day 11 compared to SIL sections, p = 0.02 com- pared to control sections, p = 0.02 at day 20 compared to SIL sections, p = 0.015 compared to control sections). When the number of cells on the articular surface was ana- lysed over time, there was no statistical difference between d11 and d20 in control or SIL sections. The number of Grap cartilage su Figure 5 h to show n rface umbers of cells observed on the articular Graph to show numbers of cells observed on the articular H and cultu extruding out of Figure 3 istologica re for 20 l section of the days showing two adjacent ch pare thent articu articular surface edge fo lar cartilage ondrocytes llowing SIL cartilage surface. It can be seen that cells were observed on Histological section of the articular surface edge following SIL the articular surface at days 11 and 20 in all three culture and culture for 20 days showing two adjacent chondrocytes conditions. At day 11 and day 20 there was a significant extruding out of the parent articular cartilage. There appears decrease in the number of cells on the cartilage surface in the to be a breach in the surface of the cartilage and cytoplasm samples cultured in the presence of 100 ng/ml BMP-2 (*) appears to be moving out of the cartilage. Stained with tolui- compared to both control and SIL sections. dine blue. ×600. Page 4 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 cells at the articular surface in SIL+BMP-2 decreased sig- nificantly between d11 and d20 (P = 0.05) Shape changes A chondrocyte was considered to have undergone a shape change when there was a clear change from the usual rounded phenotype to an elongated phenotype (Figure 6). During the culture period there was a relatively high level of shape change under all experimental conditions at all time points (Figure 7). At time = 0 there was an approx- imately 10% level of elongated cells within the cartilage in control cartilage. This was apparently increased following SIL, although this was not significant. Following culture there was no change in the numbers of elongated cells at day 11 or 20 between different treatment groups. Pyknotic cell death There was no evidence of pyknotic cell death in the Grap shape changes within the cartilage Figure 7 h to show the percentage of cells considered as having Graph to show the percentage of cells considered as having chondrocytes within the gelfoam, on the articular surface shape changes within the cartilage. It can be seen that cells or extruding from the cartilage. with an elongated phenotype were present at all time points in all three culture conditions. There was no statistically sig- Phenotypic analysis of captured cells nificant difference in cell shape between culture conditions. Immunohistochemical techniques revealed that the cap- tured cells were type I collagen negative, type II collagen and fibronectin positive and that they stained positively for proteoglycans as assessed by toluidine blue staining. In normal cartilage, at time 0, there is no evidence of any chondrocytes outside the extracellular matrix (ECM). However, outgrowth of cells into the gelatin scaffold was Discussion This study demonstrates that, following dissection dam- observed under all experimental conditions i.e. control age and/or SIL and culture, chondrocyte repair cells (no impact), SIL and SIL+BMP. extrude out from the parent cartilage and can migrate into a 3D gelatin scaffold. This sequence of events is quantita- Observation of the gelfoam at days 11 and 20 of culture tively reduced in the presence of 100 ng/ml BMP-2. revealed the presence of cells adjacent to the scaffold. In order to identify the phenotype of these cells it was neces- sary to investigate the production of ECM macromole- cules by these cells in the scaffold. This was done primarily to ascertain whether the the cells were produc- ing hyaline cartilage specific proteins i.e. to prove that they were chondrocytes. This was achieved by immun- ofluorescence techniques, using antisera that we have pre- viously demonstrated to cross react in equine cartilage. The immunofluorescence revealed that these cells were positively stained for collagen type II and fibronectin and did not stain for type 1 collagen. This indicated that the cells in the scaffold were chondrocytes and that they had no de-differentiated into fibroblasts – an important dis- tinction to make. This observation is in agreement with other authors who have demonstrated that ability of chondrocytes to retain their phenotype in 3D scaffolds [19,20]. The ability to maintain phenotype has important H p Figure 6 riesence of BMP-2 for 20 days stological section of cartilage after SIL and culture in the implications for the use of chondrocytes captured into a Histological section of cartilage after SIL and culture in the scaffold in the repair of lesions as they are continuing to presence of BMP-2 for 20 days. A normal rounded chondro- secrete a hyaline cartilage extracellular matrix which is cytes is seen (black arrow) in the same field as elongated vital for the re-formation of the articular cartilage surface. chondrocytes (red arrows). Stained with H&E. ×300. Page 5 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 As stated above, chondrocyte outgrowth into the scaffold damage in this experimental system; interestingly FGF-2 was observed in control, SIL and SIL+BMP-2 sections. has been demonstrated to induce motility in previously Therefore it can be concluded that the cartilage was stim- quiescent/non motile endothelial cells in vitro [23] and ulated sufficiently, in all of these experiments, to induce could, thus, potentially stimulate previously quiescent chondrocytes out into a gelfoam scaffold. Whilst there chondrocytes. In the experiments described in this study were moderately large numbers of cells in the gelfoam there was no appreciable difference in the numbers of cell scaffold at day 11, the number of chondrocytes in the scaf- in the gelfoam scaffold or on the cartilage surface or fold fell at day 20 indicating the cells either migrate extruding from the cartilage between control sections and through the gelfoam and are lost into the culture medium SIL sections. Thus the trigger factor for this chondrocyte or that they are dying on the scaffold. However, there was behaviour must lie outside the SIL damage in this experi- no evidence of pyknotic cell death detected histologically mental design. It has been shown that damage to cartilage among the chondrocytes in the scaffold and so migration can occur in many ways, including dissection damage (Lu out appears the most likely cause of this cell loss. et al 2006) and that such dissection damage can release growth factors such as FGF-2 (Vincent et al 2002). In this In addition to observing trends over the time course of the study it may be that release of endogenous FGF-2 is stim- experiment, the numbers of chondrocytes detected on the ulating the chondrocytes to respond and that the dissec- articular surface was also quantified in order to identify tion of the cartilage out of the joint and its handling is any differences between experimental conditions. Whilst sufficient to initiate the response. there were no significant differences between control and SIL sections, at both day 11 and day 20 there was a statis- The experiments described in this paper clearly demon- tically significant decrease in the number of cells in the strate that chondrocytes have the ability to leave the par- scaffold in SIL+BMP-2 sections. ent cartilage and migrate into an adjacent gelatin matrix following single impact load damage. This process of The exact details of the process by which the chondrocytes migration might be expected to be associated with an migrate into the scaffold is not known. However, from alteration in cell shape i.e. the cell might form a more flat- basic principles, it is clear that they must migrate out of tened, fibroblastic shape in order to move through the the parent cartilage in order to gain access to the scaffold. matrix. Our studies revealed no consistent increase in In this study we observed no evidence of cells extruding elongated cells across the experiments where the products from the cartilage at d = 0, confirming that the chondro- of migration were detected, indicating that gross shape cytes are usually confined to the ECM, as is, of course, well change to an elongated phenotype does not appear to be known. Following a period in culture we observed an indicator of motility in this model. Cell motility is a chondrocytes 'extruding' out of the articular surface i.e. complex phenomenon in which the cytoskeleton (pre- caught in the process of squeezing out of the matrix and dominately actin and vimentin) plays an essential role sitting adjacent to the articular surface. In order to investi- [24] and more detailed observations of the microstructure gate the effect of time in culture and the effect of different of the chondrocytes in this experimental model is war- culture conditions on the numbers of chondrocytes ranted. extruding from the matrix the numbers of cells extruding was quantified. In control and SIL sections there was a sta- The addition of 100 ng/ml BMP-2 to the experimental tistically significant decrease in the number of cells model significantly decreased the response of the extruding from the cartilage at day 20 compared to day 11, chondrocytes – the numbers of cells within the gelfoam, but no difference in the number of cells adjacent to the extruding and on the articular surface were reduced. The articular surface. However, there was no difference mechanism for this is not known, however, there are between control sections and those sections that had been many different ways in which BMP-2 could inhibit these SIL either in the numbers of cells on the articular surface responses. It is known that factors involved in the extru- or extruding from the cartilage. Interestingly, sections that sion and migration process of chondrocytes are the upreg- were SIL+BMP-2 had significantly less cells extruding ulation/activation of catabolic enzymes such as matrix from the matrix and sitting adjacent to the articular sur- metalloproteinases (MMPs) that degrade the matrix per- face compared to the other experimental conditions at mitting the movement of chondrocytes through. BMP-2 both day 11 and day 20. has been shown to have an inhibitory effect on the expres- sion and activity of various MMPs in a number of experi- The mechanisms driving this migration out of the parent mental situations [25,26] and it is possible that the cartilage may be due to endogenous FGF-2 release as pre- inhibitory action of BMP-2 may occur via this route. viously discussed [3,5,6]. FGF-2 has long been associated with cell motility [21,22] and could well be playing a role In our previous work we have demonstrated that FGF-2 in stimulating chondrocyte movement in response to can induce a repair response and other workers have dem- Page 6 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 onstrated the release of FGF-2 by dissection and load (Lu Competing interests et al 2006, Vincent et al 2004, 2005). If FGF-2 is driving The author(s) declare that they have no competing inter- the response of the cells out of the cartilage then it may be ests. that BMP-2 is, in some way, inhibiting at the level of FGF- 2. The individual roles played by BMP-2 and FGF-2 differ Authors' contributions in any given tissue and experimental system. However, in FH conceived the study and designed the study. TV carried some systems BMP-2 and FGF-2 are an antagonistic pair out the practical experimental work. FH drafted the man- [27]. Experiments in transgenic mice has demonstrated uscript. Both authors read and approved the final manu- that BMPs antagonize FGF signalling by inhibiting at least script. two of the intracellular pathways activated by FGFs, namely STAT and ERK. One possible way in which BMPs Acknowledgements This study was funded by the PetPlan Charitable Trust. We would like to may inhibit STAT and ERK1/2 is by negatively regulating acknowledge the Comparative Orthopaedics Research Group at the the expression of FGF signalling components [27]. Studies Department of Veterinary Medicine, University of Cambridge for their gen- have shown that BMP and FGF signalling have opposing eral contribution. actions in the growth [28], however, limb-culture studies have yielded contradictory results; some studies suggest References that BMPs exert stimulatory effects on differentiation, 1. Hunziker EB, Rosenberg LC: Repair of partial-thickness defects whilst others provide evidence to support an inhibitory in articular cartilage: cell recruitment from the synovial membrane. J Bone Joint Surg Am 1996, 78(5):721-733. effect. 2. Buckwalter JA, Mankin HJ: Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr Course Lect 1998, 47:487-504. In conclusion this study has shown that cartilage sub- 3. 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Journal of Periodontal Research 1998, 33:476-485. 27. Yoon BS, Pogue R, Ovchinnikov DA, Yoshii I, Mishina Y, Behringer RR, Lyons KM: BMPs regulate multiple aspects of growth- plate chondrogenesis through opposing actions on FGF pathways. Development 2006:4667-4678. 28. Minina E, Kreschel C, Naski MC, Ornitz DM, Vortkamp A: Interac- tion of FGF, Ihh/Pthlh, and BMP signalling integrates chondrocyte proliferation and hypertrophic differentiation. Development and Cell 2002, 3:439-449. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2474/8/120/pre pub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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Chondrocyte outgrowth into a gelatin scaffold in a single impact load model of damage/repair – effect of BMP-2

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Springer Journals
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Copyright © 2007 by Henson and Vincent; licensee BioMed Central Ltd.
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Medicine & Public Health; Orthopedics; Rehabilitation; Rheumatology; Sports Medicine; Internal Medicine; Epidemiology
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1471-2474
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10.1186/1471-2474-8-120
pmid
18053249
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Abstract

Background: Articular cartilage has little capacity for repair in vivo, however, a small number of studies have shown that, in vitro, a damage/repair response can be induced. Recent work by our group has shown that cartilage can respond to single impact load and culture by producing repair cells on the articular surface. The purpose of this study was to identify whether chondrocyte outgrowth into a 3D scaffold could be observed following single impact load and culture. The effect of bone morphogenic-2 (BMP-2) on this process was investigated. Methods: Cartilage explants were single impact loaded, placed within a scaffold and cultured for up to 20 days +/- BMP-2. Cell numbers in the scaffold, on and extruding from the articular surface were quantified and the immunohistochemistry used to identify the cellular phenotype. Results: Following single impact load and culture, chondrocytes were observed in a 3D gelatin scaffold under all culture conditions. Chondrocytes were also observed on the articular surface of the cartilage and extruding out of the parent cartilage and on to the cartilage surface. BMP-2 was demonstrated to quantitatively inhibit these events. Conclusion: These studies demonstrate that articular chondrocytes can be stimulated to migrate out of parent cartilage following single impact load and culture. The addition of BMP-2 to the culture medium quantitatively reduced the repair response. It may be that the inhibitory effect of BMP-2 in this experimental model provides a clue to the apparent inability of articular cartilage to heal itself following damage in vivo. Cartilage repair is an important clinical challenge and is Background Articular cartilage constantly experiences the damaging being studied extensively. effects of biomechanical 'wear and tear' and is reported to have little capacity for permanent repair due primarily to Work undertaken by our group has recently described a its limited proliferative capability [1]. This limited repair novel intrinsic damage repair response in mature equine capacity leads to damage accumulation, resulting in the articular cartilage explants following single impact load loss of cartilage integrity and the development of chronic and subsequent culture [3]. This response is characterised degenerative joint disease, specifically osteoarthritis (OA) by the appearance of chondrocyte repair cells on damaged [2]. cartilage. This apparent mobilisation of chondrocytes has Page 1 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 also been described following significant cartilage damage Impact loading of cartilage discs (mincing), where the chondrocytes were observed to be Discs were randomly divided into two groups – control migrating into a 3D scaffold. This scaffold was subse- (unimpacted) or impacted. Discs were impacted using a quently used to repair cartilage lesions in vivo [4]. This drop tower device following the method described previ- work demonstrates that cartilage can be stimulated suffi- ously [3,11,12]. Each disc, with the articular surface facing ciently to mobilise chondrocytes to sites of damage and down was impacted from a height of 2.5 cm using a beyond, into a scaffold, and that these mobilised cells weight of 500 g. The approximate impact energy applied have distinct and exciting clinical possibilities. to each disc was 0.175 J, impacted at a velocity of approx- imately 0.7 m/s. To ensure constant compression condi- In the damage/repair model described by our group [3] tions, the impactor was left upon the disc for 10 s before we have shown that repair cell numbers are significantly being removed. increased when the cartilage is cultured in the presence of 50 ng/ml fibroblastic growth factor-2 (FGF-2). The mech- In vitro culture of cartilage discs anism by which repair cells appear on the surface of dam- After impact, discs were placed into pockets created by aged cartilage is unknown, however we have previously sharp excision in a gelatin scaffold (Gelfoam, Pharmacia hypothesised that FGF-2 (both exogenous and from Upjohn, USA) (1 cm × 1 cm pieces). Cartilage+Gelfoam endogenous release [5,6]) is driving this process. FGF-2 is units were cultured for 0, 10 and 20 days in Dulbecco's one of a family of growth factors that have profound modified Eagle's medium (DMEM, Sigma-Aldrich, UK) effects on cartilage development and repair. Other growth supplemented with 200 IU/ml penicillin (Invitrogen, factors are also clearly involved in cartilage biology and of UK), 2.5 υg/ml streptomycin (Invitrogen, UK), 500 υg/ml these the bone morphogenic proteins (BMPs) are some of ascorbic acid (Sigma Aldrich, UK) and 10% fetal calf the most widely studied. In particular it has been shown serum (Invitrogen, UK) at 37 degrees C and 5% CO2.) that BMP-2 promotes chondrogenesis [7], induces the dif- Groups of discs were also cultured in the presence of 100 ferentiation of stem cells into chondrocytes [8] is shown ng/ml BMP-2 (Sigma Aldrich, UK). 100 ng/ml BMP-2 was to be up-regulated in cartilage after mechanical damage used in this experiment as 100 ng/ml has been shown to [9] and promotes articular cartilage repair experimentally be an effective dose for stimulation of chondrocyte and [10]. BMP-2 is therefore a growth factor of potential inter- mesenchymal stem cell responses [13,14]. Each experi- est in in vitro cartilage repair. mental time point was made up of 3 cartilage discs i.e each experiment was performed in triplicate in each of the The aims of this study were (i) to identify whether repair three animals. Control explant discs (not impacted) were cells produced following SIL and subsequent culture have cultured for the same time-periods. At the end of each the ability to migrate out of parent cartilage into a 3D gel- time point cartilage discs were embedded in Tissue Tek atin scaffold and ii) to investigate the effects of 100 ng/ml OCT (Sakura Finetek Europe, The Netherlands) and snap- BMP-2 on these events. frozen in liquid nitrogen or placed immediately into for- mal saline and paraffin embedded. Frozen sections (10 μm) were cut and placed on poly-L-lysine coated slides for Methods Horses histological and immunocytochemical analysis. Cartilage was obtained from horses aged between 7 and 9 years (n = 3) that were humanely destroyed for reasons Histological and immunocytochemical analysis other than joint disease. For this study, only normal, Sections were stained for routine histological analysis healthy cartilage was used based on the lack of pathology with Haematoxylin and Eosin (Sigma Aldrich). The fol- following macroscopical and microscopical examination. lowing features of each experimental time point were quantified cells with elongated shape, cells with pyknotic Harvesting of cartilage nuclei, cells on the surface of the section, cells extruded Cartilage discs (7 mm diam.) were dissected aseptically from the section and cells trapped in gelfoam. The quan- from the articular surface of the proximal phalanx, with- tification of each of these cell features was performed by out attached subchondral bone, using a sterile cork borer. counting 3 adjacent high power fields in the top, middle Discs were placed into sterile phosphate-buffered saline and bottom of the cartilage section using a standardised (PBS) containing antibiotics and antimycotics (200 IU/ml grid approach. penicillin, 2.5 μg/ml fungizone, 100 μg/ml streptomycin, 20 μg/ml gentamycin, Invitrogen, Paisley, Scotland), and Identification of the synthesis of hyaline cartilage specific then washed a further 3 times in sterile PBS. proteins was performed by standard immunolocalisation using the following primary antibodies – polyclonal rab- bit anti-rat collagen IX/XI (Calbiochem/Merck), polyclo- nal rabbit anti-porcine collagen type II (raised and Page 2 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 characterised by M. E. Davies), polyclonal mouse anti- bovine collagen type I(Sigma-Aldrich) and polyclonal goat anti-rabbit fibronectin (Sigma, UK). These antibod- ies have been previously shown to recognise equine epitopes ([15,16]). The appropriate FITC-conjugated sec- ondary antibodies were used to visualise the antigen. Quantitation techniques Data from all sections was pooled. Chondrocyte numbers were expressed as the percentage of cells as a total of the number of cells within the section. This calculated figure was used in order to minimise the effect of different cells numbers within sections from different animals. Statisti- cal significance was identified suing Mann-Whitney and Kruskal-Wallis tests. Results Cartilage damage following a single impact load His la Figure 1 r su tolrfa ogical s ce and the gelfoam sc ection showing th affold e junction between the articu- Cartilage discs that had been impacted suffered damage at Histological section showing the junction between the articu- the articular surface immediately upon impact. This dam- lar surface and the gelfoam scaffold. Cells have migrated out age was characterised by loss of proteoglycan (as demon- of the cartilage (bottom left of picture) and are clearly seen strated by loss of metachromatic staining), roughening of associated with the gelatin 'fibres' The arrow marks the Gel- the articular surface and fissure formation, as previously foam-cartilage junction. Stained with H&E. ×200. described ([17,18]). All control explants, in contrast, pre- sented an intact articular surface and uniform metachro- matic staining, with no apparent loss of proteoglycan. Quantification of chondrocytes in scaffold At t = 0 all chondrocytes were observed to be within the parent bone as this time point represents the intial state of the cartilage prior to culture. When cultured, chondro- cytes were observed within the gelfoam scaffold at day 11 and day 20 under all experimental conditions, including non impacted cartilage. Chondrocytes were usually observed to be closely associated with gelfoam 'fibres' sug- gesting that they may be adherent to the scaffold (Figure 1). The chondrocytes observed within the gelfoam at d11 and d 20 were quantified as a percentage of cells within the parent section. There was no significant difference between the numbers of chondrocytes in the gelfoam between control and SIL sections at d11 or d20, however there was a statistically significant difference decrease in the samples cultured with BMP-2 (p = 0.01 at day 11 com- Grap fo Figure 2 ld (h to show n 'Gelfoam') umbers of cells captured in 3D gelatin scaf- pared to SIL sections, p = 0.049 compared to control sec- Graph to show numbers of cells captured in 3D gelatin scaf- tions, p = 0.03 at day 20 compared to SIL sections, p = fold ('Gelfoam'). It can be seen that cells were detected in the 0.03 compared to control sections). When the number of Gelfoam scaffold at days 11 and 20 in all three culture condi- chondrocytes within the gelfoam at days 11 and 20 was tions. At day 11 and day 20 there were significantly reduced compared, it was shown that the percentage of chondro- numbers of chondrocytes in the Gelfoam in the samples cul- cytes in the gelfoam had significantly decreased between tured in the presence of 100 ng/ml BMP-2 (*). At day 20 from d11 to d20 in control (P = 0.005) and SIL (P = there was a significant decrease in cell numbers in all three 0.007) sections (Figure 2). There was no significant experimental conditions. change in the number of cells that had entered the gel- Page 3 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 foam scaffold from the sections supplemented with BMP- 2 over time. Cells extruded from sections Cells were considered to be extruded from the parent car- tilage if they could be observed actually exiting out of the articular surface (Figure 3). In all cases there appeared to be a breach in the articular surface through which cyto- plasm was apparently moving. Chondrocytes were observed to be extruding from the parent cartilage at days 11 and 20 under all culture conditions studied (Figure 4). At t = 0 no chondrocytes were observed to be extruding i.e. extrusion of cells from the cartilage did not appear to occur prior to culturing. There was no significant differ- ence in the number of cells extruding from the parent car- tilage in control and SIL cartilage, however, in the SIL+BMP sections there was a statistically significant decrease in the number of cells extruding at day 11 com- Grap la Figure 4 r car h to show n tilage surface umbers of cells extruding out of the articu- pared to controls (p = 0.04) and SIL sections (p = 0.05) Graph to show numbers of cells extruding out of the articu- (Figure 4). lar cartilage surface. It can be seen that cells were observed to be extruding at days 11 and 20 in all three culture condi- Cells on the articular surface tions. At day 11 there was a significant decrease in the No cells were detected on the articular surface at day 0. number of cells extruding from the cartilage surface in the Cells were detected on the articular surface at day 11 and samples cultured in the presence of 100 ng/ml BMP-2 (*) day 20 under all experimental conditions (Figure 5). compared to both control and SIL sections. There was no significant difference in the number of cells on the articular surface between control and SIL cartilage at either day 11 or 20, however the number of cells on the articular surface of the SIL+BMP-2 sections were statisti- cally decreased compared to both control and SIL, (p = 0.03 at day 11 compared to SIL sections, p = 0.02 com- pared to control sections, p = 0.02 at day 20 compared to SIL sections, p = 0.015 compared to control sections). When the number of cells on the articular surface was ana- lysed over time, there was no statistical difference between d11 and d20 in control or SIL sections. The number of Grap cartilage su Figure 5 h to show n rface umbers of cells observed on the articular Graph to show numbers of cells observed on the articular H and cultu extruding out of Figure 3 istologica re for 20 l section of the days showing two adjacent ch pare thent articu articular surface edge fo lar cartilage ondrocytes llowing SIL cartilage surface. It can be seen that cells were observed on Histological section of the articular surface edge following SIL the articular surface at days 11 and 20 in all three culture and culture for 20 days showing two adjacent chondrocytes conditions. At day 11 and day 20 there was a significant extruding out of the parent articular cartilage. There appears decrease in the number of cells on the cartilage surface in the to be a breach in the surface of the cartilage and cytoplasm samples cultured in the presence of 100 ng/ml BMP-2 (*) appears to be moving out of the cartilage. Stained with tolui- compared to both control and SIL sections. dine blue. ×600. Page 4 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 cells at the articular surface in SIL+BMP-2 decreased sig- nificantly between d11 and d20 (P = 0.05) Shape changes A chondrocyte was considered to have undergone a shape change when there was a clear change from the usual rounded phenotype to an elongated phenotype (Figure 6). During the culture period there was a relatively high level of shape change under all experimental conditions at all time points (Figure 7). At time = 0 there was an approx- imately 10% level of elongated cells within the cartilage in control cartilage. This was apparently increased following SIL, although this was not significant. Following culture there was no change in the numbers of elongated cells at day 11 or 20 between different treatment groups. Pyknotic cell death There was no evidence of pyknotic cell death in the Grap shape changes within the cartilage Figure 7 h to show the percentage of cells considered as having Graph to show the percentage of cells considered as having chondrocytes within the gelfoam, on the articular surface shape changes within the cartilage. It can be seen that cells or extruding from the cartilage. with an elongated phenotype were present at all time points in all three culture conditions. There was no statistically sig- Phenotypic analysis of captured cells nificant difference in cell shape between culture conditions. Immunohistochemical techniques revealed that the cap- tured cells were type I collagen negative, type II collagen and fibronectin positive and that they stained positively for proteoglycans as assessed by toluidine blue staining. In normal cartilage, at time 0, there is no evidence of any chondrocytes outside the extracellular matrix (ECM). However, outgrowth of cells into the gelatin scaffold was Discussion This study demonstrates that, following dissection dam- observed under all experimental conditions i.e. control age and/or SIL and culture, chondrocyte repair cells (no impact), SIL and SIL+BMP. extrude out from the parent cartilage and can migrate into a 3D gelatin scaffold. This sequence of events is quantita- Observation of the gelfoam at days 11 and 20 of culture tively reduced in the presence of 100 ng/ml BMP-2. revealed the presence of cells adjacent to the scaffold. In order to identify the phenotype of these cells it was neces- sary to investigate the production of ECM macromole- cules by these cells in the scaffold. This was done primarily to ascertain whether the the cells were produc- ing hyaline cartilage specific proteins i.e. to prove that they were chondrocytes. This was achieved by immun- ofluorescence techniques, using antisera that we have pre- viously demonstrated to cross react in equine cartilage. The immunofluorescence revealed that these cells were positively stained for collagen type II and fibronectin and did not stain for type 1 collagen. This indicated that the cells in the scaffold were chondrocytes and that they had no de-differentiated into fibroblasts – an important dis- tinction to make. This observation is in agreement with other authors who have demonstrated that ability of chondrocytes to retain their phenotype in 3D scaffolds [19,20]. The ability to maintain phenotype has important H p Figure 6 riesence of BMP-2 for 20 days stological section of cartilage after SIL and culture in the implications for the use of chondrocytes captured into a Histological section of cartilage after SIL and culture in the scaffold in the repair of lesions as they are continuing to presence of BMP-2 for 20 days. A normal rounded chondro- secrete a hyaline cartilage extracellular matrix which is cytes is seen (black arrow) in the same field as elongated vital for the re-formation of the articular cartilage surface. chondrocytes (red arrows). Stained with H&E. ×300. Page 5 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 As stated above, chondrocyte outgrowth into the scaffold damage in this experimental system; interestingly FGF-2 was observed in control, SIL and SIL+BMP-2 sections. has been demonstrated to induce motility in previously Therefore it can be concluded that the cartilage was stim- quiescent/non motile endothelial cells in vitro [23] and ulated sufficiently, in all of these experiments, to induce could, thus, potentially stimulate previously quiescent chondrocytes out into a gelfoam scaffold. Whilst there chondrocytes. In the experiments described in this study were moderately large numbers of cells in the gelfoam there was no appreciable difference in the numbers of cell scaffold at day 11, the number of chondrocytes in the scaf- in the gelfoam scaffold or on the cartilage surface or fold fell at day 20 indicating the cells either migrate extruding from the cartilage between control sections and through the gelfoam and are lost into the culture medium SIL sections. Thus the trigger factor for this chondrocyte or that they are dying on the scaffold. However, there was behaviour must lie outside the SIL damage in this experi- no evidence of pyknotic cell death detected histologically mental design. It has been shown that damage to cartilage among the chondrocytes in the scaffold and so migration can occur in many ways, including dissection damage (Lu out appears the most likely cause of this cell loss. et al 2006) and that such dissection damage can release growth factors such as FGF-2 (Vincent et al 2002). In this In addition to observing trends over the time course of the study it may be that release of endogenous FGF-2 is stim- experiment, the numbers of chondrocytes detected on the ulating the chondrocytes to respond and that the dissec- articular surface was also quantified in order to identify tion of the cartilage out of the joint and its handling is any differences between experimental conditions. Whilst sufficient to initiate the response. there were no significant differences between control and SIL sections, at both day 11 and day 20 there was a statis- The experiments described in this paper clearly demon- tically significant decrease in the number of cells in the strate that chondrocytes have the ability to leave the par- scaffold in SIL+BMP-2 sections. ent cartilage and migrate into an adjacent gelatin matrix following single impact load damage. This process of The exact details of the process by which the chondrocytes migration might be expected to be associated with an migrate into the scaffold is not known. However, from alteration in cell shape i.e. the cell might form a more flat- basic principles, it is clear that they must migrate out of tened, fibroblastic shape in order to move through the the parent cartilage in order to gain access to the scaffold. matrix. Our studies revealed no consistent increase in In this study we observed no evidence of cells extruding elongated cells across the experiments where the products from the cartilage at d = 0, confirming that the chondro- of migration were detected, indicating that gross shape cytes are usually confined to the ECM, as is, of course, well change to an elongated phenotype does not appear to be known. Following a period in culture we observed an indicator of motility in this model. Cell motility is a chondrocytes 'extruding' out of the articular surface i.e. complex phenomenon in which the cytoskeleton (pre- caught in the process of squeezing out of the matrix and dominately actin and vimentin) plays an essential role sitting adjacent to the articular surface. In order to investi- [24] and more detailed observations of the microstructure gate the effect of time in culture and the effect of different of the chondrocytes in this experimental model is war- culture conditions on the numbers of chondrocytes ranted. extruding from the matrix the numbers of cells extruding was quantified. In control and SIL sections there was a sta- The addition of 100 ng/ml BMP-2 to the experimental tistically significant decrease in the number of cells model significantly decreased the response of the extruding from the cartilage at day 20 compared to day 11, chondrocytes – the numbers of cells within the gelfoam, but no difference in the number of cells adjacent to the extruding and on the articular surface were reduced. The articular surface. However, there was no difference mechanism for this is not known, however, there are between control sections and those sections that had been many different ways in which BMP-2 could inhibit these SIL either in the numbers of cells on the articular surface responses. It is known that factors involved in the extru- or extruding from the cartilage. Interestingly, sections that sion and migration process of chondrocytes are the upreg- were SIL+BMP-2 had significantly less cells extruding ulation/activation of catabolic enzymes such as matrix from the matrix and sitting adjacent to the articular sur- metalloproteinases (MMPs) that degrade the matrix per- face compared to the other experimental conditions at mitting the movement of chondrocytes through. BMP-2 both day 11 and day 20. has been shown to have an inhibitory effect on the expres- sion and activity of various MMPs in a number of experi- The mechanisms driving this migration out of the parent mental situations [25,26] and it is possible that the cartilage may be due to endogenous FGF-2 release as pre- inhibitory action of BMP-2 may occur via this route. viously discussed [3,5,6]. FGF-2 has long been associated with cell motility [21,22] and could well be playing a role In our previous work we have demonstrated that FGF-2 in stimulating chondrocyte movement in response to can induce a repair response and other workers have dem- Page 6 of 8 (page number not for citation purposes) BMC Musculoskeletal Disorders 2007, 8:120 http://www.biomedcentral.com/1471-2474/8/120 onstrated the release of FGF-2 by dissection and load (Lu Competing interests et al 2006, Vincent et al 2004, 2005). If FGF-2 is driving The author(s) declare that they have no competing inter- the response of the cells out of the cartilage then it may be ests. that BMP-2 is, in some way, inhibiting at the level of FGF- 2. The individual roles played by BMP-2 and FGF-2 differ Authors' contributions in any given tissue and experimental system. However, in FH conceived the study and designed the study. TV carried some systems BMP-2 and FGF-2 are an antagonistic pair out the practical experimental work. FH drafted the man- [27]. Experiments in transgenic mice has demonstrated uscript. Both authors read and approved the final manu- that BMPs antagonize FGF signalling by inhibiting at least script. two of the intracellular pathways activated by FGFs, namely STAT and ERK. One possible way in which BMPs Acknowledgements This study was funded by the PetPlan Charitable Trust. We would like to may inhibit STAT and ERK1/2 is by negatively regulating acknowledge the Comparative Orthopaedics Research Group at the the expression of FGF signalling components [27]. Studies Department of Veterinary Medicine, University of Cambridge for their gen- have shown that BMP and FGF signalling have opposing eral contribution. actions in the growth [28], however, limb-culture studies have yielded contradictory results; some studies suggest References that BMPs exert stimulatory effects on differentiation, 1. Hunziker EB, Rosenberg LC: Repair of partial-thickness defects whilst others provide evidence to support an inhibitory in articular cartilage: cell recruitment from the synovial membrane. J Bone Joint Surg Am 1996, 78(5):721-733. effect. 2. Buckwalter JA, Mankin HJ: Articular cartilage: degeneration and osteoarthritis, repair, regeneration, and transplantation. Instr Course Lect 1998, 47:487-504. In conclusion this study has shown that cartilage sub- 3. 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Journal of Periodontal Research 1998, 33:476-485. 27. Yoon BS, Pogue R, Ovchinnikov DA, Yoshii I, Mishina Y, Behringer RR, Lyons KM: BMPs regulate multiple aspects of growth- plate chondrogenesis through opposing actions on FGF pathways. Development 2006:4667-4678. 28. Minina E, Kreschel C, Naski MC, Ornitz DM, Vortkamp A: Interac- tion of FGF, Ihh/Pthlh, and BMP signalling integrates chondrocyte proliferation and hypertrophic differentiation. Development and Cell 2002, 3:439-449. Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-2474/8/120/pre pub Publish with Bio Med Central and every scientist can read your work free of charge "BioMed Central will be the most significant development for disseminating the results of biomedical researc h in our lifetime." 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Published: Dec 5, 2007

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