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Rheumatology Advance Access originally published online on May 2, 2006
Rheumatology 2006 45(12):1529-1533; doi:10.1093/rheumatology/kel111
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A case of chondromatosis indicates a synovial stem cell aetiology

A. Crawford, A. Frazer, J. M. Lippitt1, D. J. Buttle2 and T. Smith3

Centre for Biomaterials & Tissue Engineering, School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield S10 2TN, 1Division of Clinical Sciences South, University of Sheffield Medical School, Royal Hallamshire Hospital, Sheffield S10 2JF, 2Section of Functional Genomics, Division of Genomic Medicine, University of Sheffield Medical School, E-Floor, Beech Hill Road, Sheffield S10 2RX and 3Department of Orthopaedic Surgery, Northern General Hospital, Sheffield S5 7AU, UK.

Correspondence to: Aileen Crawford, Centre for Biomaterials and Tissue Engineering, School of Clinical Dentistry, University of Sheffield, Claremont Crescent, Sheffield S10 2TA, UK. E-mail: a.crawford{at}sheffield.ac.uk


   Abstract
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 Abstract
Introduction
 Patient and methods
 Results
 Discussion
 References
 
Objective. To evaluate cell cultures derived from intrasynovial nodules from a patient with primary synovial chondromatosis (PSC) for aberrant numbers/differentiation of osteochondroprogenitor cells.

Methods. Cell cultures were established from PSC synovial nodules, or normal bovine or human osteoarthritis (OA) synovia (for comparison). Multi-lineage potential was determined using well-characterized in vitro culture systems to assess osteogenic, chondrogenic and adipogenic capability.

Results. Primary PSC cell cultures were typically fibroblastic but contained islands of dense cell clusters/nodules, some of which were isolated and cultured separately [putative osteochondroprogenitris (pOCP) cultures]. OA synovial cultures had barely detectable levels of alkaline phosphatase (AP) that increased (0.006±0.008 to 0.141±0.000 nmol p-nitrophenol/min/cm2) with dexamethasone treatment. AP activity was higher in primary PSC cell cultures and further enhanced by dexamethasone (from 0.076±0.022 to 5.735±0.000 nmol p-nitrophenol/min/cm2). Histochemically, AP was localized as discreet areas within PSC cultures. No AP activity was detected histochemically in OA or normal bovine synovial cultures. The pOCP cultures had high basal AP (5.036±0.439 nmol p-nitrophenol/min/cm2) and spontaneously formed mineralized nodules, which increased in number under standard osteogenic conditions. Under chondrogenic conditions, micromass or pellet-cultured pOCP cells spontaneously synthesized a matrix containing glycosaminoglycans and collagen II. In adipogenic medium, the number of lipid-containing cells was increased.

Conclusions. Compared with cultures established from OA or normal synovia, cell cultures established from PSC synovial nodules were enriched in osteochondroprogenitors, which, unlike normal mesenchymal cells, differentiated along chondrogenic and osteogenic lineages in the absence of dexamethasone.

KEY WORDS: Primary synovial chondromatosis, Disease aetiology, Stem cells, Osteochodroprogenitors, Mesenchymal progenitors, Chondrogenesis, Osteogenesis.


   Introduction
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 Abstract
 Introduction
Patient and methods
 Results
 Discussion
 References
 
Primary synovial chondromatosis (PSC) is a rare disorder. Although the aetiology is unknown, it is accepted that the condition has a proliferative component [1] suggestive of a benign metaplasia of the synovium. However, the phenotype and origin of the cells causing the metaplasia and production of multiple intrasynovial cartilaginous nodules remain to be unknown. One possibility is a mesenchymal progenitor cell origin. Mesenchymal progenitor cells (MPCs) have a high proliferation potential and can differentiate into several mesenchymal lineages including cartilage, bone, tendon, ligament and adipocytes [2]. MPCs are found in low numbers in osteoarthritic synovial fluid [3] and normal human synovium, and are induced in vitro to differentiate along chondrogenic, osteogenic and adipogenic lineages [4]. Recently, fibroblast growth factor 3-receptor (FGFR3), a marker of cartilage progenitors and mesenchymal stem cells, was observed on a limited number of cells within PSC synovium and superficial layers of cartilage nodules, and its ligand, FGF9, was present in PSC but not in normal synovial fluid [5]. Our objective was to determine whether intrasynovial cartilaginous nodules from a patient with primary synovial chondromatosis (PSC) may have had a stem cell origin. We investigated this by assessing whether cell cultures isolated from synovial nodules had enhanced multi-lineage capability compared with cultures established from normal bovine or human OA synovia.


   Patient and methods
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 Abstract
 Introduction
 Patient and methods
Results
 Discussion
 References
 
A male nurse, now 42 years old, first presented in 1992 with pain and swelling of the knee. A loose body was identified radiologically and removed arthroscopically. At arthroscopy, no OA was evident. Synovial inflammation was noted but a biopsy showed ‘non-specific inflammatory changes’. In 1994, a definite effusion was noted, and CT and X-ray examination showed evidence of synovial chondromatosis in the region of the infra-patellar fat pad. This was excised and histology confirmed typical changes of synovial chondromatosis with proliferation of the synovium and calcification of multiple intra-synovial cartilaginous nodules. Further pain and swelling of the knee occurred and X-ray examination showed typical changes of synovial chondromatosis in the posterior aspect of the joint. A localized synovectomy was performed and, as before, there was gross thickening of the synovium with calcified, cartilaginous nodules in large numbers in the affected area. PSC was confirmed histologically and samples of the abnormal tissue were taken for cell isolation and histology.

Synovial tissue was also collected from two female patients with osteoarthritis, aged 67 and 69 years at operation for knee replacement. All patient tissues were collected with informed consent and ethical approval.

Cell culture
PSC nodules were digested (2 h, 37°C) with a mixture of bacterial collagenase (Clostridium histolyticum, Sigma, 2 mg/ml) and neutral protease (Bacillus polymyxa, Sigma, 1 mg/ml). The isolated cells were cultured as monolayers (PSC cultures) in Dulbecco's Modification of Eagle's Medium containing non-essential amino acids, 10 mM Hepes buffer, 10% fetal calf serum and 25 µg/ml L-ascorbic acid (basic medium). Samples of bovine or human osteoarthritic synovium were treated in the same way. Confluent cultures were passaged using a non-enzymic, cell-dissociating medium (Invitrogen) according to the manufacturer's instructions. Cell cultures at passage 2–3 were used for experimentation.

Cell nodules that formed in PSC monolayers were isolated using cloning rings and non-enzymic cell dissociating medium. Cells from several nodules were combined and cultured in basic medium to yield cultures referred to as putative osteochondroprogenitors (pOCP). The numbers of pOCP cells were expanded in monolayer culture and used at passage 5–17 for experimentation.

In vitro osteogenesis assays
Osteogenesis was performed as described previously [6, 7]. Monolayers were incubated in basic medium containing 10–8 M dexamethasone then basic medium containing 10–8 M dexamethasone and 10 mM ß–glycerophosphate. Alkaline phosphatase activity was quantified using p-nitrophenol phosphate (1 mM, n ≥ 3) and the enzyme was localized in cell cultures with naphthol phosphate AS-BI (50 µg/ml) and fast red (1 mg/ml). Mineralized nodules were stained with alizarin red (0.1%, pH 5.5, 30 min).

In vitro chondrogenesis assays
Chondrogenesis was assessed as described previously [8, 9]. Cell pellets (2 x 105 cells/pellet) or micromass cultures (4 x 105 cells/micromass) of pOCP cells were incubated in serum-free basic medium containing 10 µg/ml insulin, 5.5 µg/ml transferrin, 4.7 µg/ml linoleic acid, 5 ng/ml sodium selenate, 1.75 mg/ml bovine serum albumin, with or without 10–7 M dexamethasone, and 10 ng/ml transforming growth factor ß1 (TGFß1). After 21 days, pellet cultures were harvested for quantitative determination of sulphated glycosaminoglycans (GAGs) [10] or embedded in optimal cutting temperature, OCT, cryo-embedding agent for immunohistochemical detection of collagen I and II [11]. Micromass cultures [4] were incubated for 7 or 14 days. After 7 days, micromasses were fixed with methanol and stained with alcian blue (1% in 1N HCl, pH 1.0) to detect GAGs. After 14 days, micromasses were fixed with 4% paraformaldehyde and collagens I and II were visualized immunochemically [11].

Adipogenesis assay
Confluent cultures of pOCP cells were treated with adipogenic induction medium [4] and then maintained for 5 days in basic medium containing 10 µg/ml insulin. They were fixed with 2% glutaraldehyde and stained with oil red O solution to detect triglyceride accumulation.


   Results
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 Abstract
 Introduction
 Patient and methods
 Results
Discussion
 References
 
Histology and cell culture
Excised tissue showed a typical pathology of PSC [12] with dense cartilaginous and calcified nodules embedded within a hyperplastic synovium.

Cells from bovine or OA synovial tissue exhibited a spindle-shaped, fibroblastic morphology typical of synovial fibroblasts. Cell cultures derived from PSC synovial nodules contained predominantly spindle-shaped, fibroblastic cells typical of synovial fibroblasts. However, these cultures (PSC) also contained discreet, raised cell clusters or nodules containing cells of rounded morphology (Fig. 1a). Isolation and culture of the cell clusters yielded monolayer cultures (pOCPs) with a polygonal morphology and rapid proliferation (i.e. pOCPs reached confluency within 3–4 days compared with 2–3 weeks for OA or bovine synoviocytes). At confluence, the pOCP cells exhibited a cobblestone appearance with dense nodular cell clusters.


Figure 1
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  		FIG. 1. (a) Nodule formation in PCS synovial cell cultures (Scale bar = 200 µm). (b) Alkaline phosphatase localization in confluent monolayer OA (A, B) and PCS (C, D) synovial cultures with (B, D) and without (A, C) 10–8 M dexamethasone for 14 days. (c) In vitro osteogenic assay on pOCP cultures. (A, control; B, treated with 10–8 M dexamethasone, then 10–8 M dexamethasone plus 10 mM ß-glycerophosphate; C, treated with 10 mM ß-glycerophosphate only; D, treated with 10–8 M dexamethasone only).

 
In vitro osteogenesis
Dexamethasone enhanced alkaline phosphatase activity in the PSC synovium cultures (0.076±0.022 to 5.735±0.000 nmol p-nitrophenol/min/cm2). The enzyme was localized as discreet areas within the cultures (Fig. 1b), suggesting a sub-population of cells (pOCP) had differentiated in response to dexamethasone. However, the steroid did not induce nodule formation nor enable mineralization in the PSC synovium-derived cultures in the presence of ß-glycerophosphate. OA synoviocyte cultures had virtually undetectable alkaline phosphatase activity that increased with dexamethasone (0.006±0.008 to 0.141±0.000 nmol p-nitrophenol/min/cm2). No histochemical staining for alkaline phosphatase was detected in normal bovine (results not shown) or OA synoviocyte cultures (Fig. 1b) suggesting a low level of osteochondroprogenitor cells in these cultures. Neither nodule formation nor mineralization was induced by dexamethasone in bovine or OA synoviocyte cultures.

Confluent pOCP cultures expressed high alkaline phosphatase activity and formed nodules, some of which mineralized on addition of ß-glycerophosphate (Fig. 1c); indicating spontaneous osteogenic differentiation. In common with its effect on MSC differentiation, dexamethasone increased alkaline phosphatase activity in pOCP cultures (5.036±0.439 to 7.087±0.156 nmol p-nitrophenol/min/cm2) and promoted the number of nodules. Addition of ß-glycerophosphate with dexamethasone increased the number of mineralized nodules (Fig. 1a), whereas alkaline phosphatase in these mineralized cultures was reduced (5.432±1.400 nmol p-nitrophenol/min/cm2), indicating mature mineralized nodules [6].

In vitro chondrogenesis
pOCP pellet and micromass cultures without dexamethasone spontaneously produced a matrix containing sulphated GAGs, collagen II and barely detectable collagen I (Fig. 2a and b); all features characteristic of a chondrocytic phenotype. Collagen II deposition was also observed in cultures treated with dexamethasone and/or TGFß1. Addition of TGFß1 alone, or with dexamethasone, promoted collagen I accumulation in pellet and micromass cultures, but had no effect on the level of matrix GAGs compared with control (results not shown). However, dexamethasone alone reduced matrix GAGs (8.05±2.11 to 4.71±1.31 µg/pellet). In pellets treated with TGFß1 plus dexamethasone, the peripheral third of the cells were of elongated appearance (results not shown). Deposition of collagen I, lack of stimulation of proteoglycan accumulation and induction of a fusiform morphology suggested that TGFß1 and dexamethasone promoted differentiation of the pOCP cells towards a hypertrophic chondrocyte phenotype [13].


Figure 2
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  		FIG. 2. Chondrogenic and adipogenic potential of nodule-derived pOCP cells. Cells were incubated as micromass cultures in chondrogenic medium. (a) Collagens I and II were detected by immunohistochemistry after 14 days. NS are controls for non-specific staining. (Treatments were A, dexamethasone 10–7M and TGFß 10 ng/ml; B, TGFß 10 ng/ml, C, dexamethasone 10–7 M, D, medium only control.) (b) GAGs were detected with alcian blue (treatments A to D as for Fig 2a). (c) Monolayers of pOCP cells were treated with control (plate A) or lipogenic medium (plate B) and stained with oil red O to detect lipid (Scale bar = 25 µm).

 
In vitro adipogenesis
Control pOCP cultures contained few lipid-containing cells, but their numbers increased markedly on exposure to adipogenic induction medium (Fig. 2c).


   Discussion
Top
 Abstract
 Introduction
 Patient and methods
 Results
 Discussion
References
 
Using in vitro culture systems, we investigated the multi-lineage potential of cells derived from intrasynovial cartilaginous nodules of a patient with primary synovial chondromatosis. Primary cultures (PSC) contained dense cell clusters which were isolated and cultured separately (pOCP cultures). The marked rise in alkaline phosphatase of dexamethasone-treated PSC cultures indicated that they contained putative osteoprogenitors which differentiated to an osteogenic phenotype with dexamethasone. Histochemical localization of alkaline phosphatase in the PSC monolayers showed a sub-population of cells which differentiated in response to dexamethasone. Dexamethasone also promoted alkaline phosphatase activity in OA synovial cell cultures, but to a lower extent than the PSC cultures and was not detected histochemically. This suggested an enrichment of osteochondroprogenitors in the PSC cultures compared with normal bovine or OA synovium cultures.

Under appropriate conditions in vivo [14, 15] or in vitro [8, 16], MPCs proliferate and differentiate to hypertrophic chondrocytes, osteoblasts and adipocytes. pOCP cells were highly proliferative, formed nodules and spontaneously differentiated along chondrogenic and osteogenic pathways, and were induced to differentiate along an adipocyte lineage. Hence, the pOCP cells were multipotent and may have caused the cartilaginous and mineralized nodules observed in the PSC patient. In contrast, MPCs from normal synovium required dexamethasone to initiate differentiation [4].

OA synovial tissue was collected from two female patients who were older than the PSC patient. It is possible that age may influence the number of putative MSCs present in normal synovium. However, neither basal nor dexamethasone-induced alkaline phosphatase was detected in bovine synoviocytes from skeletally mature, young animals, indicating few MPCs in the bovine cultures. Obtaining normal human synovium was not ethically feasible.

Our findings from one PSC patient could suggest a deregulation of MPC differentiation in joints affected by PSC. The aetiology of highly proliferative osteoprogenitor cells with a predilection to differentiate abnormally in a restricted number of articular joints in this patient remains unknown, but may involve a somatic mutation in a key gene governing MPC differentiation. Recently, it was reported that a deregulation of hedgehog signalling (due to a mutation in the target gene, Gli3) is a predisposition for synovial chondromatosis [17]. Increased cell proliferation may result from increased FGF2 secretion by the PSC nodule cells. Evidence for the presence of FGF2 and its receptor FGFR1 in synovial chondromatosis (and absence in OA) was recently reported [18].

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Patient and methods
 Results
 Discussion
 References
 

  1. Davis RI, Foster H, Arthur K, Trewin S, Hamilton PW, Biggart DJ. (1998) Cell proliferation studies in primary synovial chondromatosis. J Pathol 184:18–23.[CrossRef][ISI][Medline]
  2. Caplan AI. (1991) Mesenchymal stem cells. J Orthop Res 9:641–50.[CrossRef][ISI][Medline]
  3. Jones EA, English A, Henshaw K, et al. (2004) Enumeration and phenotypic characterisation of synovial fluid multipotential mesenchymal progenitor cells in inflammatory and degenerative arthritis. Arthritis Rheum 50:817–27.[CrossRef][ISI][Medline]
  4. De Bari C, Dell';Accio F, Tylzanowski P, Luyten FP. (2001) Multipotent mesenchymal stem cells from adult human synovial membrane. Arthritis Rheum 44:1928–42.[CrossRef][ISI][Medline]
  5. Robinson D, Hasharoni A, Evron Z, Segal M, Nevo Z. (2000) Synovial chondromatosis: the possible role of FGF9 and FGF receptor 3 in its pathology. Int J Exp Path 81:183–89.[CrossRef][ISI][Medline]
  6. Maniatopoulos C, Sodek J, Melcher AH. (1988) Bone formation in vitro by stromal cells obtained from bone marrow of young adult rats. Cell Tissue Res 254:317–30.[ISI][Medline]
  7. Malaval L, Modrowski D, Gupta AK, Aubin JE. (1994) Cellular expression of bone-related proteins during in vitro osteogenesis in rat bone marrow stromal cell cultures. J Cell Physiol 158:555–72.[CrossRef][ISI][Medline]
  8. Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. (1998) In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 238:265–72.[CrossRef][ISI][Medline]
  9. Yoo JU, Barthel TS, Nishimura K, et al. (1998) The chondrogenic potential of human bone marrow-derived mesenchyma progenitor cells. J Bone Joint Surg 80-A:1745–57.[Abstract/Free Full Text]
  10. Farndale RW, Buttle DJ, Barrett AJ. (1986) Improved quantitation of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta 883:173–77.[Medline]
  11. Kafienah W, Jacob M, Demarteau O, et al. (2002) Three-dimensional tissue engineering of hyaline cartilage: comparison of adult nasal and articular chondrocytes. Tissue Eng 8:817–26.[CrossRef][ISI][Medline]
  12. Milgram JW. (1977) Synovial osteochondromatosis: a histopathological study of thirty cases J Bone Joint Surg 59-A:792–801.
  13. Kergosien N, Sautier J, Forest N. (1998) Gene and protein expression during differentiation and mineralisation in a chondrocyte cell culture system. Calcif Tissue Int 62:114–21.[CrossRef][ISI][Medline]
  14. Ashton BA, Allen TD, Howlett CR, Eaglesom CC, Hatton A, Owen M. (1980) Formation of bone and cartilage by marrow stromal cells in diffusion chambers in vivo. Clin Orthop 151:294–307.
  15. Haynesworth SE, Goshima J, Goldberg VM, Caplan AI. (1992) Characterisation of cells with osteogenic potential from human marrow. Bone 13:81–88.[Medline]
  16. Asahina I, Sampath TK, Nishimura I, Hauschka PV. (1993) Human osteogenic protein-1 induces both chondroblastic and osteoblastic differentiation of osteoprogenitor cells derived from newborn rat calvaria. J Cell Biol 123:921–33.[Abstract/Free Full Text]
  17. Hopyan S, Nadesan P, Wunder J, Alman BA. (2005) Disregulation of hedgehog signalling predisposes to synovial chondromatosis. J Pathol in press.
  18. Sato J, Segami N, Suzuki T, Yoshitake Y, Nishikawa K. (2002) The expression of fibroblast growth factor-2 and fibroblast growth factor receptor-1 in chondrocytes in synovial chondromatosis of the mandibular joint. Report of two cases. Int J Maxillofac Surg 31:532–36.[CrossRef]
Submitted 22 June 2004; revised version accepted 28 February 2006.
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This Article
Right arrow Abstract Freely available
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