Fibrocytes Participate in the Development of Heterotopic Ossification Abelardo Medina, MD, PhD, FACS,*‡ Zengshuan Ma, PhD,* Mathew Varkey, PhD,* Hongbin Liu, MD,‡ Takashi Iwashina,‡ Jie Ding, MD, PhD,* Edward E. Tredget, MD, MSc, FRCSC*†‡

Heterotopic ossification (HO) is a complication of musculoskeletal injury characterized by the formation of mature bone in soft tissues. The etiology of HO is unknown. We investigated the role of bone marrow derived progenitor cells in HO pathophysiology. We isolated the cells from HO specimens by cell explantation. Using flow cytometry and immunofluorescence microscopy, we found that 35 to 65% of the HO cells exhibit a bone marrow derived fibrocyte profile consisting in spindle-shaped morphology associated with type 1 pro-collagen and LSP1 expression. When cultured in osteogenic differentiation medium, active machinery for bone mineralization (high gene expression of Anx2, TNAP, and Pit-1), and calcium/phosphate deposits were found. Interestingly, interferon-alpha 2b significantly reduced the proliferation rate and COL1 gene expression in HO cells. We have characterized a novel subset of bone marrow derived progenitor cells in the HO specimens. The findings from this research study will provide new insights into the development of HO in burn patients. (J Burn Care Res 2015;36:394–404)

Heterotopic ossification (HO) is a clinical entity characterized by the formation of mature lamellar bone in damaged tissues such as muscle, fascia, and tendon.1 Genetic disorders (ie, fibrodysplasia ossificans progressive), traumatic injuries (ie, brain injury, spinal cord injury, burns, and amputations) and musculoskeletal surgeries (ie, hip arthroplasty, elbow/ acetabular fractures), among others, have been associated with this condition.1–5 The management of HO lesions is still controversial and with variable levels of success due to, at least in part, their pathophysiology is not well understood.6–8 It seems that dysregulation of local and systemic factors work in concert to create a permissive milieu for ectopic bone formation. In

this regard, the growth of HO lesions requires both local upregulation of fibrogenesis to synthesize new unmineralized matrix and the presence of bone forming cells to produce the progressive mineral nucleation of this scaffold.9 Based on these facts, it is our hypothesis that the recruitment of circulating bone marrow derived cells with potential for osteoblastic differentiation may contribute to exacerbate and/or perpetuate an osteoinductive environment. The study of these recruited bone marrow derived cells may provide new insights into not only the pathophysiology of heterotopic ossification formation, but also information for the development of novel therapeutic strategies to control their progression.

From the *Wound Healing Research Group, Division of Plastic and Reconstructive Surgery, Department of Surgery, University of Alberta, Edmonton, Canada; †Division of Critical Care Medicine; and ‡Firefighters’ Burn Treatment Unit, Department of Surgery, University of Alberta, Edmonton, Canada. Address correspondence to Edward E. Tredget, MD, MSc, FRCSC, University of Alberta, 2D2.28 WMC, 8440-112 Street, Edmonton, Alberta, Canada, T6G 2B7. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site. Copyright © 2014 by the American Burn Association 1559-047X/2015

METHODS

DOI: 10.1097/BCR.0000000000000102

394

Collection of Tissue Samples Heterotopic ossification specimens were collected during the surgical release of affected joints from three patients admitted to the Firefighters’ Burn Treatment Unit of the University of Alberta Hospital (Edmonton, Canada). Patients provided written consent prior to the surgery. The Health Research Ethics Board of the University of Alberta approved the protocols used in this report.

Journal of Burn Care & Research Volume 36, Number 3

Isolation and Culture of Cells From Heterotopic Ossification Specimens Cells were isolated by cell explantation after the HO specimens were washed several times in sterile 1X phosphate buffered saline (PBS). Several small tissue fragments were put on petri dishes and cultured in Dulbecco’s modified Eagle medium (DMEM, GibcoLife Technologies Inc, Carlsbad, CA, USA) + 10% fetal bovine serum (FBS, Gibco) + streptomycin sulfate (100 μg/ml) + penicillin G sodium (100 units/ ml) and amphotericin B (0.25 μg/ml) (Gibco) at 37 °C, 95% humidity, and 5% carbon dioxide (see Figure, Supplemental Digital Content 1 at http://links.lww. com/BCR/A20). Subsequently, the confluent cells were harvested with trypsin/ethylenediaminetetra acetic acid (EDTA) (Gibco) for 5 minutes at 37 °C and subcultured into 75 cm2 flasks (BD Biosciences, San Jose, CA, USA). To induce proliferation, the cells were cultured in similar conditions in proliferative medium (PM) as described above, which were changed every 48 hours. To stimulate the osteogenic differentiation, the cells were cultured in 89% low-glucose DMEM + 10% fetal bovine serum (FBS) + 1% antibiotics associated with 100 nM dexamethasone (Sigma-Aldrich; St Louis MO, USA), 50 to 80 μg/ml of L-ascorbic acid 2-phosphate (ASAP; Sigma-Aldrich) and 10 mM beta-glycerophosphate (Sigma-Aldrich). This osteogenic differentiation medium (ODM) was changed every 48 hours.

Flow Cytometry Analysis To prevent nonspecific binding and background fluorescence, 100 μl of Fc blocking agent (diluted in Fluorescence-activated cell sorting (FACS) buffer at 1:50 ratio; BD Biosciences) was added to each sample and incubated on ice for 20 minutes. The cells were then permeabilized with 0.5% saponin in 1X PBS and incubated with mouse anti-human primary antibodies for type 1 pro-collagen (COL-1; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and rabbit anti-human leukocyte specific protein 1 (LSP-1; Abnova, Walnut, CA, USA). Primary antibody solutions were applied to 100 μl of cell suspension in permeabilization solution. The corresponding isotype non-immune IgGs were used as controls. After incubation for 1 h on ice, the samples were washed thrice with washing solution (100 ml 1X PBS + 100 μl of Tween-20). Subsequently, fluorescent-conjugated secondary antibodies were applied to cell suspensions and incubated on ice for 45 to 60 minutes in dark conditions. Thus, APC CY7-conjugated goat anti-mouse IgG1 (Santa Cruz Biotechnology) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG (Life Technologies Inc.) were

Medina et al   395

used. Double staining with type 1 pro-collagen and Leukocyte-specific protein 1 (LSP1) was considered a distinctive phenotype to isolate the fibrocyte population as previously described.10 To identify the expression of alpha smooth muscle actin (α-SMA), the cells were incubated with PEconjugated mouse anti-human α-SMA antibody (R&D Systems, Minneapolis, MN) before 10,000 cells were analyzed on a FACS Canto machine (BD Biosciences) using DiVa software (BD Biosciences).

Immunofluorescence and Immunocytochemistry Microscopy The cells from HO specimens, cultured on chambered slides (Nunc Lab-Tek II Chamber Slide System, Thermo Scientific, Pittsburgh, PA, USA), were fixed in 4% paraformaldehyde for 10 minutes, and then washed in 1X PBS and graded ethanol at room temperature. Non-specific bindings were avoided by treating the cells with a blocking solution (1X PBS containing 10% goat serum + 5% bovine serum albumin; Sigma-Aldrich; St Louis MO, USA). For immunofluorescence, the primary antibody rat anti-human N-terminal propeptide of type 1 collagen (MAB1912; EMD Millipore Corporation, Billerica, MA, USA) was applied and incubated for 1 hour. After two washing steps with PBS-Tween 20 for 5 minutes each, the samples were incubated with rhodamine-conjugated goat anti-rat IgG F(ab’)2 fragment (AP136R; MD Millipore Corporation) for 45 minutes in a dark condition. Subsequently, the samples were washed with PBS-Tween 20 and followed by the incubation (1 hour) with the second primary antibody, mouse anti-human LSP-1 mAb (BD Transduction Laboratories, Franklin Lakes, NJ, USA). After washing with PBS-Tween 20 twice for 5 minutes each, the samples were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG F(ab’)2 fragment (AP181F; EMD Millipore Corporation) for 45 minutes in a dark condition. Finally, the samples were mounted in ProLong® Gold Antifade Reagent with DAPI (Molecular Probes-Life Technologies Inc, Carlsbad, CA, USA). An Olympus BX40 microscope was utilized with the software Image-Pro plus (MediaCybernetics). For immunocytochemistry, endogenous peroxidases were inactivated with 30% hydrogen peroxide (SigmaAldrich) diluted in methanol (2% vol/vol) at room temperature for 15 minutes. After several washing steps with Double-distilled water (ddH2O) and rehydration with 1X PBS for 5 minutes, the samples were incubated in blocking solution (1X PBS + 10% goat serum + 5% bovine serum albumin [Sigma-Aldrich]) in a humidified chamber for 30 minutes to prevent non-specific



Journal of Burn Care & Research May/June 2015

396   Medina et al

bindings. Rabbit anti-human alpha-smooth muscle actin monoclonal antibody (EMD Millipore Corporation, Billerica, MA, USA) was added and incubated in a humidified chamber overnight at 4 °C. After washing with 1X PBS (thrice for 5 minutes each), the secondary antibody goat anti-rabbit IgG (Lot BA-9200/ Vector Laboratories Inc.) was added and incubated for 45 to 60 minutes at room temperature. Subsequently, the application of StreptoABComplex/HRP was carried out according to manufacturer recommendations (Dako Cytomation, DK-2600 Glostrup, Denmark). The samples were then exposed to DAB solution ([3, 3’-Diaminobenzidine [Sigma-Aldrich] 25 mg + 1X PBS 50 ml + 30% hydrogen peroxide 50 μl). This peroxidase substrate was applied until the samples acquired slight brown staining. Counterstaining with haematoxylin was additionally performed utilizing a standard protocol. Dehydration using graded ethanol and treatment with xylene were performed before the application of Permount (Fisher Scientific; Fair Lawn, New JerseyUSA) and coverslips. Images were obtained using a Zeiss Axioplan 2 imaging microscope with Northern Eclipse image analysis software.

Masson’s Trichrome Staining HO specimens were sectioned at a thickness of 5 microns, mounted on slides and fixed with 10% formalin. Subsequently, the slides were stained with Weigert’s iron haematoxylin solution for 10 minutes. After the washing step, the slides were stained with Biebrich scarlet-acid fuchsin solution for 15 minutes followed by phosphomolybdic-phosphotungstic acid solution for 15 minutes. The slides were then transferred into aniline blue solution for 5 to 10 minutes. After treatment with in 1% acetic acid solution for 5 minutes and rinsing in distilled water, the sections were quickly dehydrated in 95% ethyl alcohol and anhydrous ethyl alcohol, and then cleared in xylene before the tissue sections were covered by Permount (Fisher Scientific) and slide coverslips.

Liquid Chromatography/Mass Spectrometry analysis The 4-hydroxyproline content in conditioned media was measured to quantify the production and release of collagen to the extracellular space by cultured HO cells. The culture media were changed to 1 ml of DMEM containing ascorbic acid (50 μg/ml), βaminopropionitrile (50 μg/ml), proline (0.1 mM/L; 0.0115 mg/ml), and 2% FBS. After 48 hours in culture, the conditioned media were collected to precipitate the existing collagen with acetonitrile, and subsequently centrifuge them at 4 °C for 15 minutes. The precipitates were hydrolyzed in 6 N hydrochloric acid solution

at 110 °C overnight. After drying, a known amount of N-methyl-proline was added to the hydrolysate to obtain the N-butyl ester derivative of hydroxyproline. Liquid chromatography/mass spectrometry analysis was performed on an HP1100 LC linked to a HP 1100 Mass Selective detector monitoring the ions 188 (N-butyl ester of 4-hydroxyproline) and 186 (N-butyl ester of N-methyl-proline). Each sample was run in triplicate and the results are displayed as nanograms of 4-hydroxyproline per 105 cells per 48 hours obtained by reference to a standard curve of 4-hydroxyproline analyzed under the identical conditions and the results are presented as mean ± standard error. Wells with HO conditioned medium alone were set up as controls. Mass spectrometry was also used to identify the total proteins released by HO cells. After several washings with 1X PBS, the cells were set in culture using DMEM without FBS at 37 °C for 48 hours. Subsequently, the resulting supernatants containing products released by HO cells (~60 ml) were concentrated to a volume of ~600 μl by using 100 kDa MWCO Centricon Plus-20 centrifugal filter devices (EMD Millipore Corporation). Thirty micrograms of total proteins were loaded in a ready 12% Tris-HCl precast gel for polyacryamide electrophoresis (#161– 1102; Bio-Rad, Hercules, CA, USA). After running the gels, the resulting bands (2 mm) were excised and subjected to trypsin digestion. LC-MS/MS work was performed on an LTQ Orbitrap XL (Thermo Scientific). The data was processed and peptide sequences were searched against the Uniprot database using Proteome Discoverer 1.2 (Thermo scientific).

Total Cell Count In order to quantify the proliferation rate, HO cells from different individuals were separately cultured in 6-well plates (BD Biosciences) at 37 °C, 95% humidity, and 5% carbon dioxide. Twenty thousands cells were added to each well and cultured with PM, which were changed every 48 hours. After two weeks in culture, the cells were washed thrice with 1X PBS and harvested by using 0.1% trypsin (Gibco) + 0.05% EDTA (Gibco) in 1X PBS at 37 °C for 5 minutes. After neutralization (DMEM + 10% FBS), the cells were washed several times with 1X PBS. Once in suspension, the total number of cells for each well was calculated using a manual cell counter and phase contrast microscopy.

3-(4,5-Dimethyl-2-Thiazolyl)-2,5-Diphenyl2H-Tetrazolium Bromide Reduction Assay (MTT) for Cell Proliferation Assay Similar experimental setting as above (total cell count) was prepared for MTT assay. In this procedure, the

Journal of Burn Care & Research Volume 36, Number 3

yellow tetrazole MTT (3-(4,5-dimethyl-2-thiazolyl)2,5-diphenyl-2H-tetrazolium bromide) is metabolically reduced by living cells. The resulting intracellular purple formazan can be quantified as indirect expression of cell proliferation rate. Thus, to carry out this experiment, the MTT powder was dissolved in sterile 1X PBS, pH 7.4 at a concentration of 5 mg/ml, and subsequently filtered through a 0.22 μm filter. After the removal of the culture medium or specific treatment (see below), 500 μl of MTT solution was added to each well. The plates were wrapped with aluminum foil and incubated at 37 °C, 95% humidity, and 5% carbon dioxide for 5 hours. After removing the MTT solution and washing with 1X PBS, 1 ml of dimethyl sulfoxide (DMSO; Sigma-Aldrich) was added to each well and the plates were shaken for 20 minutes to dissolve formazan crystals. Immediately after this, the plates were transferred to a microplate reader (Molecular Devices, Sunnyvale, CA, USA) to measure the absorbance (optical density) of the resulting solution in each well at 570 nm. MTT assays were performed in triplicate.

Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR) To detect and quantify the variations on gene expression of crucial factors involved in osteogenesis and mineralization processes, the HO cells were cultured to 80 to 100% confluence in 6-well plates using ODM. After treatment, the cells were collected by TRIzol reagent (Invitrogen-Life Technologies Corporation) for RNA extraction. The total RNA was extracted using RNeasy Mini Kit (Qiagen Sciences Inc., Germantown, MD, USA). A 0.5 μg total RNA was used to complementary DNA (cDNA) synthesis using cDNA Syntheses Kit (Invitrogen-Life Technologies Corporation). Real-time RT-PCR was conducted using Power SYBR Green PCR Master Mix (Applied Biosystems-Life Technologies Corporation) in a 25 μl volume containing 5 μl of 1:10 diluted cDNA product and 1 μM primers listed in Table 1. Human hypoxanthine phosphoribosyltransferase 1 (HPRT1) was used as a normalization standard (Eurofins MWG/Operon, Huntsville, AL, USA). Amplification and analysis of the cDNA fragments were carried out by StepOnePlus RT-PCR System (AB Applied Biosystems-Life Technologies Corporation). The relative expression of genes of interest was measured as cycle threshold and normalized with individual HPRT1 control cycle threshold values.

Alizarin Red S Staining This experimental procedure was used to ­identify the presence in intracellular calcium deposits. After

Medina et al   397

treatment with ODM for five days, the cells were washed several times with 1X PBS and fixed in 10% buffered formalin at room temperature for 45 minutes. After fixation, the cells were rinsed with ddH2O, and subsequently stained with 2% alizarin Red S (Sigma-Aldrich) in water for 10 minutes. After rinsing several times with ddH2O, the slides were allowed to air dry before the cells were examined under the microscope. Cells treated with PM were used as the control. The images of cells were taken by a microscope Nikon-Optiphot 2 (Nikon Instruments Inc., Melville, NY, USA).

Von Kossa Staining The precipitation reaction induced by this technique allows the detection of intracellular phosphate deposits. After treatment with ODM for five days, the cells were washed several times with 1X PBS and fixed with 4% paraformaldehyde at room temperature for 20 minutes. After fixation, the cells were rinsed with ddH2O, and subsequently incubated with 1% silver nitrate solution while treated with ultraviolet light for 20 minutes. Then, the cells were washed several times with ddH2O and incubated with 5% sodium thiosulfate for 3 minutes to remove residual silver. Finally, the cells were rinsed again in ddH2O, and quickly dehydrated in consecutive treatments with 70%, 95%, and 100% alcohol (3 minutes each). The images were taken by a microscope Nikon-Optiphot 2 (Nikon Instruments Inc).

Interferon Alpha 2b Treatment to Control Proliferative Capacity In this experimental setting, the HO cells were cultured in 6-well plates at 20,000 cells per well. Cells from all the wells were maintained in culture with PM, which was replaced every 48 hours. Treatment with interferon-α2b at 3,000 units/ml daily was used to interrupt the cell cycle progression, and reduce the proliferative capacity of HO cells. After two weeks, the treated and control (untreated) groups were harvested as described above. To further confirm the effect of interferon-α2b on proliferation of HO cells, the total cell count and MTT assay were performed.

Statistical Analysis Non-parametric tests were used to determine the statistical significance of the results. Specifically, MannWhitney rank sum test was performed to evaluate independent sample medians using GraphPad InStat 3 (GraphPad Software Inc, La Jolla, CA, USA) for windows. The results were presented as mean ± SEM, where a P ≤ .05 was considered statistically significant.



Journal of Burn Care & Research May/June 2015

398   Medina et al

Table 1. Nucleotide sequences of real-time reverse transcription-polymerase chain reaction (RT-PCR) primers Gene COL1A1 MMP1 Osteocalcin Osterix Anx A2 TNAP Pit-1 HPRT1

Forward Primer

Reverse Primer

5’-GCC TCG GAG GAA ACT TTG C-3’ 5’-TCG CTG GGA GCA AAC ACA-3’ 5’-TGA GAG CCC TCA CAC TCC TC-3’ 5’-TGG GCA AAG CAG GCA CAA AG-3’ 5’-CTC TAC ACC CCC AAG TGC AT-3’ 5’-CGG GCA CAA TGA AGG AAA A-3’ 5’-TAC AAC TCG ACT CAA GGG CTA CTG-3’ 5’-CGG CTT GCT CGA GAT GYG AT-3’

RESULTS HO Lesions Exhibit a Fibrotic Architecture The process of ectopic bone formation in soft tissues starts as localized increase of density, which progressively incorporates more mineralized deposits (Figure 1A). HO lesions are usually located in injured tissues around major joints (especially hip and elbow) producing a significant reduction of their range of motion necessary to perform activities of daily living (Figure 1A). At advanced HO stages, surgical excision is the only reliable method to improve the function of affected joints as illustrated

5’-TCC GGT TGA TTT CTC ACT ATA GC-3’ 5’-TTG GCA AAT CTG GCG TGT AA-3’ 5’-CGC CTG GGT CTC TTC ACT AC-3’ 5’-GGG AAT GAG TGG GAA AAG GGA G-3’ 5’-TCA GTG CTG ATG CAA GTT CC-3’ 5’-GCC AGA CCA AAG ATA GAG TT-3’ 5’-GGG GAC ACG AAC CAA GAC ATC ACA-3’ 5’-GCA CAC AGA GGG CTA CGA TGT-3’

in Figure 1B. Interestingly, HO specimens display not only osteogenic profile, but also a vascularized fibrotic structure. Thus, Masson’s trichrome stains showed dense collagenous scaffold surrounding HO cells (Figure 1C).

Recruitment of Circulating Bone MarrowDerived Cells to HO Lesions We isolated and characterized HO cells using cell explantation and flow cytometry, respectively. Interestingly, flow cytometric analysis showed that up to 65% of the explanted cells from HO specimens exhibit double staining for LSP1 and type 1

Figure 1.  Heterotopic ossification in a burn patient. The left upper image of (A) shows no bony or other joint abnormalities seen 40 days after burn injury. The right upper image of (A) reveals a significant increase in the heterotopic ossification (HO) lesion (5 months after burn injury). This patient had 11° of preoperative range of motion (ROM) obtained from −55° of maximal extension (left lower image [A]) and 66° of maximal flexion (right lower image [A]). (B) shows the surgical removal of HO lesion by posteromedial longitudinal incision and the immediate improvement of the ROM (69° added to the ROM). (C) shows the HO specimen (left image) and the subsequent Masson’s trichrome staining that reveal the high density of collagenous scaffold in the HO structure. Scale bars: 100 and 20 μm.

Journal of Burn Care & Research Volume 36, Number 3

Medina et al   399

Figure 2. Identification of bone marrow-derived precursor cells in Heterotopic ossification specimens. Flow cytometric analysis revealed that between 35 and 65% of the cells present in heterotopic ossification (HO) specimens exhibit a fibrocyte profile with a distinctive double positive staining for LSP1 and type 1 pro-collagen (A). Immunofluorescence microscopy confirmed the co-localization of these two markers in spindle-shaped fibroblast-like cells, the typical morphology of fibrocytes in culture (B). Scale bar: 100 μm.

procollagen, a distinctive fibrocyte profile as we have previously described (Figure 2A). HO cells were also negative for CD14, CD68, CD86, and CD204 (data not shown). Immunofluorescence microscopy confirmed the presence of fibrocytes as spindle-shaped fibroblast-like cells that colocalize LSP1 and type 1 procollagen (Figure 2B). The fibrocyte profile was further established with the detection of fibronectin, vimentin, entactin, aminopeptidase N (CD13), and

TIMPs in the mass spectrometry analysis of conditioned media released by these cells (Table 2). The results suggest that HO lesions contain an important population of recruited bone marrowderived blood-borne cells. The fact that these cells displayed fibrocyte phenotype also suggests that they may play a role in the production of new unmineralized organic matrix required for the deposition of new bone materials, and consequently for the HO

Table 2. List of selected proteins released by fibrocyte-like cells from heterotopic ossification (HO) lesions Accession

Protein Name

MW (kDa)

Score

N

SC (%)

Functions

P02751 P08670

Fibronectin Vimentin

262.4 53.6

181.88 992.22

13 16

9.51 38.63

P14543 P15144 P02452

Entactin Aminopeptidase N COL1A1

136.3 109.7 138.9

22.26 17.29 205.26

5 3 8

7.94 7.14 7.99

P01033

TIMP1

23.2

380.45

5

44.44

Catalytic activity, protein binding, structural molecule activity Catalytic activity, motor activity, protein binding structural molecule activity Metal ion binding, protein binding Catalytic/receptor activity, signal transducer Protein binding, structural molecule activity, transcription regulator activity Enzyme regulator, metal ion binding, protein binding



400   Medina et al

Figure 3. Heterotopic ossification (HO) cells release new unmineralized organic matrix to the extracellular space. As hydroxyproline is essentially restricted to collagen, hydroxyproline content assay was used to quantify the amount of collagen produced and released by HO cells. Thus, HO cells exhibited a large release of newly formed collagen into the conditioned media, which was significantly higher when compared to that in fibroblasts (P value: .05). The negative control corresponds to HO cells in culture without the components of hydroxyproline content assay.

growth. In this regard, hydroxyproline content assay demonstrated that these cells have high capacity to synthesize collagen, which is actively released into the conditioned media (Figure 3). This result was statistical significant with a P value of .005.

Bone Marrow-Derived HO Cells Contribute to Ectopic Bone Formation We were interested to determine whether these cells also participate in the bone mineralization process. By using real-time RT-PCR, these cells showed upregulation of genes associated with osteogenesis such as osteocalcin and osterix (Figure 4A). Furthermore,

Journal of Burn Care & Research May/June 2015

these cells exhibited high gene expression of key components of the machinery required to reach adequate ion composition (calcium and phosphate) in the extracellular space for mineralization. For instance, genes such as annexin A2 (AnxA2), tissue nonspecific alkaline phosphatase (TNAP) and type III Na-Pi transporter (Pit-1) were up-regulated (Figure 4B). To further establish their bone-forming commitment, the cells were cultured under ODM. Thus, alizarin red S staining and Von Kossa staining confirmed the presence of intracellular deposits of calcium and phosphate, respectively (Figure 4C). By using flow cytometry, it was confirmed that these cells maintained the positive double staining for LSP1 and type 1 procollagen when they concluded the osteogenic differentiation process (data not shown).

Osteogenic Differentiation Inhibits Cell Proliferation of HO Cells The presence of an osteoinductive environment as seen in HO sites induces cells to progress into not only the mineralization process, but also into an irreversible differentiation pathway. HO cells have high proliferation rate doubling their population in approximately 72 hours (data not shown). However, we found that HO cells in culture displayed an increasing expression of alpha-smooth muscle actin (α-SMA; Figure 5A). Accordingly, the presence of α-SMA in HO cells treated with ODM was corroborated by immunocytochemistry (Figure 5B). Simultaneously, HO cells in ODM exhibited a decrease in their proliferative capacity. Thus, both the total cell count and MTT assay demonstrated a significant lower proliferation rate in HO cells treated with

Figure 4.  Bone marrow-derived heterotopic ossification (HO) cells contribute to ectopic bone formation. Bone marrow-derived cells in HO specimens exhibited an osteogenic commitment. (A) shows that HO cells up-regulated the gene expression of osteocalcin and osterix. They also up-regulated crucial genes associated with the machinery required for the intracellular mineralization such as Anx A2, tissue nonspecific alkaline phosphatase (TNAP) and Pit-1 (B). In addition, when the HO cells are in osteogenic differentiation medium (ODM), alizarin red S, and Von Kossa stainings demonstrated the intracellular deposits of calcium and phosphate, respectively (C). Scale bar: 20 μm.

Journal of Burn Care & Research Volume 36, Number 3

Medina et al   401

Figure 5.  Osteogenic differentiation inhibits cell proliferation of heterotopic ossification (HO) cells. The use of osteogenic differentiation medium (ODM) stimulates HO cells to express α-SMA, a well-known marker for terminal differentiation of fibrocytes. Accordingly, this finding was confirmed by flow cytometry (A) and immunocytochemistry (B). Scale bars: 20 and 100 μm. Simultaneously, ODM decreased the proliferative capacity in HO cells. Thus, both total cell counts and MTT assays demonstrated a significant lower proliferation rate in HO cells treated with ODM compared to those treated with standard proliferative medium (PM). This difference was statistically significant with a (*) P value of .05 (C).

ODM compared to those treated with standard PM. This difference was statistically significant with a P value of .05 (Figure 5C).

Interferon-α2b Interferes With Proliferative Capacity of HO Cells Based on previous findings, we targeted cell proliferation to control HO growth. This therapeutic approach may be applicable in early stages of HO to inhibit the disease progression or after surgical excision of HO lesions to prevent recurrences (Figure 1). Thus, the use of interferon alpha 2b (IFN-α2b) was able to attenuate the proliferation rate in HO cells (Figure 6A). After two weeks of treatment, both the total cell count and MTT assay demonstrated lower

presence of cells in those treated with IFN-α2b compared to those untreated (Figure 6B). The results were statistically significant with P values of .05 and

Fibrocytes participate in the development of heterotopic ossification.

Heterotopic ossification (HO) is a complication of musculoskeletal injury characterized by the formation of mature bone in soft tissues. The etiology ...
1MB Sizes 2 Downloads 4 Views