The Role of Chondrocytes in Intramembranous and EndochondralOssification During Distraction Osteogenesis in the Rabbit

G. Li, 1* A. H. R. W. Simpson,1 J. T. Triffitt 2

1The Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford, OX3 7LD, UK2MRC Bone Research Laboratory, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford, UK

Received: 13 March 1997 / Accepted: 22 September 1998

Abstract. We have used a rabbit leg-lengthening model fordetailed studies of the histology of distraction osteogenesis.Some unusual features of the endochondral ossification thatoccurs during the rapid transition of cartilage to bone in theregenerate were observed. Histological staining techniquestogether with immunohistochemistry and nonradioactiveinsitu mRNA hybridization for cartilage and bone-relatedmolecules have been used to document the presence of anoverlapping cartilage-bone phenotype in cells of the carti-lage-bone transitional region. In those particular areas, somechondrocytes appeared to be directly transformed intonewly formed bone trabeculae which are surrounded bybone matrix. Acid phosphatases were found within the car-tilage matrix in some of the cartilage/bone transitional re-gions and type I collagen mRNA and type II collagen pro-tein were found together in some of the marginal hypertro-phic chondrocytes. This study indicates an unusual role ofchondrocytes in the process of ossification at a distractionrate of 1.3 mm/day in the rabbit. Further direct evidence isrequired to prove the hypothesis that the hypertrophic chon-drocytes may transdifferentiate into bone cells in this model.

Key words: Hypertrophic chondrocyte — Transdifferen-tiation — Distraction osteogenesis — Rabbit model — Col-lagen —In situ hybridization — Immunohistochemistry.

Induction of osteogenesis by means of an osteotomy, fol-lowed by fixation with an external fixator and subsequentcontrolled distraction of the callus, is a developing tech-nique with widespread clinical application in the treatmentof bone defects, limb deformities, and fracture nonunions[1, 2]. Previous reports on distraction osteogenesis at lowrates of distraction (0.5–1.0 mm/day) in the human [3, 4],dog [5–7], and sheep [8, 9] show that the bone forms byintramembraneous ossification and no endochondral ossifi-cation is observed. However, in the rabbit model of leglengthening, direct cartilage formation from the central fi-brous tissue of the regenerate is observed [10, 11].

We have used a rabbit leg-lengthening model for de-tailed studies of the histology of the tissues formed by dis-traction osteogenesis. Using specific matrix stains (alcianblue/sirius red) [12], enzyme detection (acid phosphatase),

immunohistochemical detection of type II collagen protein,and the detection of the mRNA expression of type I colla-gen gene byin situ hybridization technique, we have ob-served some unusual morphological features of the endo-chondral ossification that occurs during the transition ofcartilage to bone in distraction osteogenesis.

Materials and Methods

Animal Model of Leg Lengthening

Six New Zealand white rabbits (aged 24 weeks, body weight 3.0–3.5 kg) were anesthetized by intramuscular injection of Hypnorm(0.2 ml/kg) (Janssen Animal Heath, High Wycombe, England),intravenous injection of hypnovel (2 mg/kg), Roche, Welwyn Gar-den City, England), and locally infiltrated with 0.25% Marcain(Astra Pharmaceutical Ltd, England). A 4-cm incision was madeover the medial aspect of the left tibia, and an Orthofix M-100lengthener (Orthofix, Italy) was fixed with four stainless-steelscrews inserted in the tibia. The tibia was osteotomized at thetibiofibular junction between the two inner screws using a handsaw under saline irrigation. Seven days after surgery, the tibia waslengthened at a rate of 1.33 mm/day by turning the lengthener byone turn (0.67 mm linear displacement) every 12 hours for 15days. Weekly radiography confirmed the lengthening rate.

Sample Preparation and Histological Examination

The rabbits were killed by anesthetic overdose at the end of theexperiment and the central portion of the distraction gap, the re-generate, was excised and fixed in 4% (w/v) buffered paraform-aldehyde for 24 hours before decalcification in neutral EDTA(14.5%, pH 7.2) at room temperature for 14–30 days. The regen-erate was embedded in paraffin wax and 5mm sections were cut bymicrotome and placed on poly-L-lysine-(Sigma, Poole, UK)coated slides for histology and immunohistochemistry or on3-aminopropyltriethoxy silane-(Sigma) coated slides forin situmRNA hybridization. The sections were dewaxed with xylene andrehydrated in four sequential ethanol baths from 99% to 50%ethanol, each for 5 minutes. For histological examination, the sec-tions were stained with hematoxylin and eosin (H&E staining).Weigerts hematoxylin/alcian blue/sirius red stain [12] was used todistinguish bone matrix (red) from cartilage matrix (blue).

Tartrate-Resistant Acid Phosphatase Staining

For identifying bone-resorbing cells, tartrate-resistant acid phos-phatase (TRAP) staining was performed. In brief, paraffin sectionswere dewaxed and rehydrated through ethanol, washed in Tris-HCL buffer (pH 9.0), and incubated with acetate buffer (0.5% w/wsodium acetate, 0.83% v/v acetic acid, pH 5.0) for 1 hour. The

Correspondence to:G. Li at his present address*Present address:The Department of Trauma and OrthopaedicSurgery, Queen’s University of Belfast, Musgrave Park Hospital,Belfast, BT9, 7JB, UK

Calcif Tissue Int (1999) 64:310–317

© 1999 Springer-Verlag New York Inc.

substrate mixture consisted of 15 mg Naphthol AS B1 phosphate(Sigma), 75 mg sodium tartrate, 1 mg pararosaniline, and 0.8 mgsodium nitrite in 26 ml acetate buffer (pH 5.0). The substratemixture was filtered before use and sections were incubated withthe mixture at 37°C for 1 hour. Sections were rinsed in runningwater for 5 minutes and counterstained by Gill’s hematoxyline(Sigma) for 15 seconds, and mounted in aquamount (BDH, Poole,England).

Immunohistochemistry of Type II Collagen

Specific goat antibodies to rabbit type II collagen [13] were usedfor immunohistochemistry. In brief, the sections were rehydratedin tris-buffered saline, pH 7.3, for 15 minutes at room temperatureand incubated in 2% hyaluronidase (ovine testicular, Sigma) inphosphate-buffered saline (PBS) for 1 hour at 37°C. They werethen washed in PBS and incubated with heat-inactivated normalrabbit serum for 15 minutes at room temperature. The specific goatanti-rabbit type II collagen antibodies was applied at a dilution of1:40 in PBS and incubated overnight at 4°C. After washing, thesections were incubated with rabbit anti-goat IgG conjugated withalkaline phosphatase for 1 hour at room temperature. Sectionswere then treated with visualizing substrates, X-phosphate, andNBT mixture according to the manufacturer’s instruction (Boeh-ringer-Mannheim, East Sussex, UK) for 30 minutes. The sectionswere then washed in PBS and mounted in glycerine jelly. Controlsections were taken by the same procedure but without the primaryantibodies.

In Situ Hybridization of Type I Collagen mRNA

The messenger RNA (mRNA) for type I collagen was detected bynucleic acid hybridization. Riboprobes for type I collagen mRNAwere made from a cDNA probe, pHCAL1U (gift from E. Vuorio,University of Turku, Turku, Finland). To generate the antisenseprobe, the plasmid was linerized with HindIII and incubated with

T7 polymerase; for the sense probe, it was linerized with EcoRIand incubated with SP6 polymerase. The riboprobes were labeledwith digoxygenin according to the protocols provided with thelabeling kit (Boehringer Mannheim, GmbH, Germany).

The protocol for in situ hybridization was as described bySandberg and Vuorio [20] with some modifications. Briefly, therehydrated sections were pretreated with 0.2 N hydrochloric acidfor 20 minutes at room temperature before incubation with pro-teinase K at 20mg/ml in 0.1 M Tris buffer (pH 8.0) and 50 m MEDTA for 10 minutes at 37°C. This was followed by fixation in4% paraformaldehyde in PBS for 20 minutes on ice and washingwith 7.5 mg/ml of glycine in PBS for 20 minutes. The tissues werethen acetylated in 0.25% acetic anhydride in 0.1 M triethanolamine(pH 8.0) for 10 minutes. The sections were dehydrated throughgraded alcohol and dried sections were then hybridized with asolution consisting of 50% formamide, 10 mM Tris-HCl (pH 7.4),1.0 mM EDTA, 0.02% polyvinyl pyrrolidone ficoll, 0.02% bovineserum albumin, 0.5% sodium dodecyl sulfate, and digoxygenin-labeled probe; 40ml of the mixture was applied to each slide andcovered with coverslip. After overnight incubation at 55°C, thecoverslips were removed with 2 × SSC (standard saline citrate) andthe sections were washed twice with 0.5 × SSC (55°C, 5 minuteseach) and 0.1 × SSC at 55°C for 5 minutes. Anti-digoxygeninantibody was incubated with the sections at room temperature for1 hour prior to development of the color by alkaline phosphatasesubstrates (Boehringer Mannheim, East Sussex, UK) for 2–4 hoursin the dark. No counterstaining was used and the sections weremounted in glycerine jelly.

Results

The regenerate contains a central fibrous zone, mainly fi-broblastic cells; the new bone developed from the centralfibrous zone and extended to both osteotomized ends. Thenewly formed bone was highly oriented toward the distrac-

Fig. 1. Representative of morphological appearance of the distraction regenerate. The regenerate contains a central fibrous zone (f) andmoderate amounts of cartilage (arrows). Alcian blue and sirius red staining, ×8.

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tion forces (Fig. 1). Moderate amounts of cartilage withinthe regenerate were found and the cartilage arose directlyfrom the central fibrous tissue in some regions (Fig. 2A),extending to new bone trabeculae without distinct bound-aries (Fig. 2B). Cartilage remnants were often seen in thenew bone trabeculae, where chondrocytes were buried into

bone matrix, as demonstrated by alcian blue and sirius redstaining (Fig. 3A, B). TRAP-positive cells were seen inmost areas of the cartilage-bone junction and the TRAP-positive staining was also observed in the cartilage matrix inareas of cartilage-bone transitional interface (Fig. 3C).

Small areas of cartilage were also found adjacent to the

Fig. 2. (A) H & E staining showing the cartilage developing fromthe central fibrous tissue. Note typical endochondral ossification atthe cartilage/bone transitional region. ×100.(B) High power view

of the cartilage/bone transitional regions. Some chondrocytes re-main intact in their lacunae within the new woven bone trabeculae(arrows). H & E staining, ×400.

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Fig. 3. (A) Chondrocytes transforminginto new bone trabeculae withsurrounding bone matrix.(B) In areas ofcartilage-bone transition, chondrocyteswere seen directly transforming into newbone matrix. b: new bone; c: cartilage; H:haversian cannel. A and B, alcian blueand sirius red staining, ×100.(C)Tartrate-resistant acid phosphatase(TRAP) staining at the cartilage-bonetransitional region showing staining in thecartilage matrix (arrows), ×100.

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periosteum, where cartilage was developing from the peri-osteal cells to form an intermediate step in new bone for-mation at the site. In some bone-cartilage transition regions,cells with chondrocyte morphology appeared to transform

into new bone trabeculae (Fig. 4A).In situ hybridizationwith type I collagen mRNA antisense probe showed that thecells with the strongest expression of type I collagen mRNAwere localized on the new bone surfaces; these cells were

Fig. 4. (A) H&E staining shows the region where cartilage wasdeveloping from fibrous tissue. Note the cartilage remnants in thenew bone trabeculae (arrow).(B) In situhybridization showing thetype I collagen mRNA present in the newly forming chondrocytes

at the subperiosteal region, and diminishes as the chondrocytesdifferentiate further and became hypertrophic. Type I collagenmRNA is also detected in the cartilage remnants within the newtrabeculae (arrow). A and B, ×100.

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flattened and were cuboidal multilayered preosteoblasts orosteoblasts. A few newly formed osteocytes in the core ofbone trabeculae expressed type I mRNA also, but the signalwas weaker and diminished as the woven bone completedcalcification (Figs. 4B and Fig. 5A). In the region of fi-brous-cartilage transition, the cartilage developed from thefibrous tissue which expressed type I collagen mRNA (Fig.4A and Fig. 5A). The type I collagen mRNA still remainedin the newly forming chondrocytes in the transitional mar-gin between the fibrous tissue and the cartilage. With furtherdifferentiation of the chondrocytes, type I collagen mRNAexpression was greatly reduced and the chondrocytes be-came hypertrophic (Figs. 4B and Fig. 5A). At the transi-tional region between cartilage and bone, type I collagenmRNA was detected in a small number of cells within thehypertrophic chondrocyte region (Fig. 5B). In addition toinsitu hybridization with type I collagen mRNA probe, im-munohistochemistry using specific antibody to type II col-lagen was applied to adjacent sections; the presence of typeII collagen protein in these cells confirmed that the marginalcells between cartilage and bone were indeed chondrocytesand that they were expressing type I collagen mRNA (Fig.6A–C).

Controls for type II collagen immunostaining were per-formed without applying primary antibody. Hybridizationusing the sense probe of collagen type I was used as control

for thein situhybridization procedure. The controls were allnegative, with only minimum background staining observed(not shown).

Discussion

Previous studies on distraction osteogenesis in human, dog,and sheep species were carried out at rates of distractionbetween 0.5 and 1.0 mm/day. Bone formation by intramem-braneous ossification was observed to be the unique char-acteristic of distraction osteogenesis in these species. How-ever, in rabbits, cartilage was observed during tibial orfemoral lengthenings by using Orthofix unilateral lengthen-ers at rates of 0.5–1.0 mm/day [10, 11]. This difference maybe due to species differences in response to distraction or itmay be a result of instability of the fixed osteotomy.

In the present study, we have used a rabbit model of leglengthening similar to that described by Kojimoto et al. [10]and found that moderate amounts of cartilage are present inthe distraction regenerate, with a mixture of endochondraland intramembraneous ossification occurring. Resultant in-stability of the osteotomy at this distraction rate (1.3 mm/day) cannot be ruled out and the cartilage formation maysuggest interference with the blood supply to the regener-ating tissues at the relatively high rate.

Fig. 5. (A) In situ hybridization with type Icollagen antisense mRNA probe. The type Icollagen mRNA is strongly expressed by thechondrocytes at the fibrous-cartilage transitionalregion (arrow), the expression diminishes as thechondrocytes differentiated and becamehypertrophic, and type I collagen mRNA isdetected again in a few hypertrophicchondrocytes at the cartilage-bone transitionalregion (B). F: fibrous tissue; H: hypertrophicchondrocytes; ×100.(B) Close-up of the boxedarea in(A) Type I collagen mRNA is presentin a few hypertrophic chondrocyte lacunae(arrows), ×400.

G. Li et al.: Role of Chondrocytes in Distraction Osteogenesis 315

Two histological staining techniques, immunohisto-chemistry and nonradioactivein situ hybridization, havebeen used to document the presence of an overlapping car-tilage/bone phenotype in cells of the cartilage/bone transi-tion region. In these region, we observed that some chon-drocytes appeared to be directly transformed into newlyformed bone trabeculae surrounding by bone matrix. Thiswas clearly shown by alcian blue/sirius red staining and thisis termed “chondroid bone” [14]. When the undifferentiatedosteogenic progenitor cells in the newly formed skeletaltissue are stimulated to differentiate, some do not go downa simple pathway to an osteoblast or chondrocyte. Becauseof their environment and systemic and local factors, somecells may appear to become “ambivalent” and may becomehybrid cells producing chondroid bone or fibrocartilage,

which may produce matrices with both types I and II col-lagens (D. Ashhurst, personal communication).

An interesting observation was the presence of acidphosphatase within the cartilage matrix in the cartilage/bonetransition region. Similar observations have been reportedby Scammell and Roach [15] in a study of fracture healingin the rabbit, although the contribution of enzyme diffusionduring reaction procedure has not been completely investi-gated. They found acid phosphatase activity within intactchondrocytic lacunae and they defined this as “lacunar”bone. They suggest a role for chondrocytes in fracture re-pair, with endochondral ossification, including direct boneformation by former chondrocytes. Robles-Marin et al. [16]reported an increase of acid phosphatase activity in thechondrocytes next to the vascular invasion during endo-chondrial ossification. They proposed that vascular invasionwill occur only after the secretion of acid phosphatases fromthe chondrocyte into the cartilage matrix. Disintegration ofthe cartilage matrix in the distraction regenerate is proposedto occur by oxidation following vascularization.

At the cartilage-bone transitional regions, type I collagenmRNA and type II collagen protein were found together insome of the marginal hypertrophic chondrocytes. This couldbe a result of direct transformation of hypertrophic chon-drocytes into osteoblasts or a result of invasion of the chon-drocyte lacunae by osteoblast progenitor cells, but it is im-possible to distinguish between these two hypotheses fromthe current study. Previous studies of chondrocyte differen-tiation have indicated that hypertrophic chondrocytesinvitro may express characteristic proteins for the osteoblastphenotype [4]. Expression of these proteins by hypertrophicchondrocytes has also been observedin vivo in selectedcartilage regions.In situ hybridization and/or immunohis-tochemistry shows that human, rat, and chicken hypertro-phic chondrocytes may be capable of synthesizing of type Icollagen [8, 17, 18, 19, 20, 21], osteocalcin [18, 21], osteo-pontin [22], and osteonectin [23, 24]. In addition, Galotto etal. [25] have shown that preosteoblasts and osteocytes sharethe same membrane protein as hypertrophic chondrocytes.Nevertheless, none of these published observations conclu-sively prove that hypertrophic chondrocytes give rise toosteoblasts directly.

An alternative explanation for the presence of type Icollagen mRNA in the chondrocytic lacunae is that theselacunae may be invaded by other cells outside the plane ofsection. They are likely to be osteogenic progenitor cellsbrought into the chondrocyte lacunae by the invading bloodvessels.

In conclusion, morphological observations, togetherwith the pattern of acid phosphatase activity, expression oftype I collagen mRNA, and localization of type II collagenprotein, indicate a process of bone ossification via cartilageprogenitors at higher rates of distraction in the rabbit. Theterminal fate of hypertrophic chondrocytes is still contro-versial and it has been suggested that it may be different atdifferent microanatomical sites [26]. Previous reports re-garding chondrocyte and osteoblast turnover, together withthe present findings, suggest that osteoblasts and chondro-blasts originate from a common pool of mesenchymal cells.These differentiated cells could share some plasticity ofphenotype. However, in this report, as in all other studiespublished to date, more direct evidence is required beforeany claims can be made that the hypertrophic chondrocytesmay transform into bone-forming cells.

Acknowledgments.The authors thank Dr. E. Vuorio for the gen-erous gifts of type I collagen cDNAs; Dr. A. S. Virdi and Dr. D. E.

Fig. 6. (A) H&E staining shows that the cartilage (arrow) is pre-sent between fibrous tissue and new bone.(B) Immunostainingusing anti-rabbit type II collagen antibody. The chondrocytes at thetransitional region are synthesizing type II collagen proteins (ar-row). (C) In situ hybridization using type I collagen antisenseprobe. Type I collagen mRNAs are detected in the chondrocytes atthe upper layer of the cartilage-bone transitional region (emptyarrow), but not at the middle layer of the cartilage (solid arrow). b:bone; c: cartilage; f: fibrous tissue. A, B, and C, ×100.

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Ashhurst for helpful advice and discussion. Dr. G. Li is supportedby a K. C. Wong scholarship at the University of Oxford.

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