Abstract
Background:
Heterotopic ossification frequently develops following high-energy blast injuries sustained in modern warfare. We hypothesized that differences in the population of progenitor cells present in a wound would correlate with the subsequent formation of heterotopic ossification.
Methods:
We obtained muscle biopsy specimens from military service members who had sustained high-energy wartime injuries and from patients undergoing harvest of a hamstring tendon autograft. Plastic-adherent cells were isolated in single-cell suspension and plated to assess the prevalence of colony-forming cells. Phenotypic characteristics were assessed with use of flow cytometry. Individual colony-forming units were counted after an incubation period of seven to ten days, and replicate cultures were incubated in lineage-specific induction media. Immunohistochemical staining was then performed to determine the percentage of colonies that had differentiated along an osteogenic lineage. Quantitative real-time reverse-transcription polymerase chain reaction was used to identify changes in osteogenic gene expression.
Results:
Injured patients had significantly higher numbers of muscle-derived connective-tissue progenitor cells per gram of tissue (p < 0.0001; 95% confidence interval [CI], 129,930 to 253,333), and those who developed heterotopic ossification had higher numbers of assayable osteogenic colonies (p < 0.016; 95% CI, 12,249 to 106,065). In the injured group, quantitative real-time reverse-transcription polymerase chain reaction performed on the in vitro expanded progeny of connective-tissue progenitors demonstrated upregulation of COL10A1, COL4A3, COMP, FGFR2, FLT1, IGF2, ITGAM, MMP9, PHEX, SCARB1, SOX9, and VEGFA in the patients with heterotopic ossification as compared with those without heterotopic ossification.
Conclusions:
Our study suggests that the number of connective-tissue progenitor cells is increased in traumatized tissue. Furthermore, wounds in which heterotopic ossification eventually forms have a higher percentage of connective-tissue progenitor cells committed to osteogenic differentiation than do wounds in which heterotopic ossification does not form. The early identification of heterotopic ossification-precursor cells and target genes in severe wounds not only may be an effective prognostic tool with which to assess whether heterotopic ossification will develop in a wound, but may also guide the future development of individualized prophylactic measures.
Level of Evidence:
Prognostic Level III. See Instructions to Authors for a complete description of levels of evidence.
Wound-healing requires the orchestration and orderly progression of a number of cellular and molecular events1. Impairment of the normal wound-healing process may lead to a delay in wound closure and aberrant tissue repair responses. Heterotopic ossification, defined as the formation of mature bone in non-osseous tissue, can develop in traumatic wounds2,3. Heterotopic ossification most commonly occurs as a sequel of head trauma, blunt elbow or acetabular trauma, and burns4-8 and following total hip arthroplasty9,10.
Modern blast injuries are often characterized by prolonged systemic inflammation and devastating injury patterns, involving massive zones of injury7,11-13, as up to 80% of survivors of combat-related injuries sustain major extremity trauma. Heterotopic ossification occurs in approximately 64% of these patients, and at least 19% of those who develop heterotopic ossification require additional surgical procedures for excision of the heterotopic ossification7,11, with postoperative wound complication rates as high as 24%13. The occurrence of heterotopic ossification in the military population results in substantial morbidity and prolonged rehabilitation. An Injury Severity Score of ≥16, definitive amputation within the zone of injury, and a blast mechanism of injury were found to be significant risk factors for the development of heterotopic ossification (p < 0.05), suggesting that both systemic and local factors may play a role in triggering the onset of heterotopic ossification11,14,15.
The mechanism(s) involved in formation of heterotopic ossification have not been fully elucidated. It is thought that the formation of heterotopic ossification requires an inciting injury that creates a conducive tissue microenvironment consisting of cellular and molecular signaling events that support the differentiation of endogenous and/or recruited precursor cells with the potential to differentiate into cartilage or bone2. With respect to progenitor cells, mesenchymal stem cells are characterized as undifferentiated cells that support the normal repair and regeneration of damaged tissue. Recently, Nesti et al.16 reported the presence of multipotent mesenchymal stem cells in war wounds. In a subsequent study, their group proposed that mesenchymal stem cells are the putative cells in the development of heterotopic ossification17. This hypothesis was based on an in vitro comparison demonstrating increased osteogenic activity in mesenchymal stem cells derived from wounds of injured soldiers and bone-marrow-derived mesenchymal stem cells harvested from patients undergoing total hip replacement. Similarly, adult periosteal cells have shown multilineage potential, given the proper induction signals, both in vivo and in vitro18 and have been considered to be essential cells in the development of heterotopic ossification. These recent studies implicating mesenchymal stem cells in the development of heterotopic ossification are important in that they established the presence of a multipotent cell in the anatomic region at risk following blunt trauma. However, the findings of those studies were based on the osteogenic properties of serially cultured cells, which may not reflect the behavior of osteogenic precursor cells in vivo. Several authors have reported that ex vivo expansion of human mesenchymal stem cells can lead to a decrease in the osteogenic differentiation capacity of the cells as well as their general multipotency19-21.
We hypothesized that there is a greater number of assayable muscle-derived connective-tissue progenitors following severe combat-related trauma and that these progenitors have a measurable osteogenic potential.
Subjects
The study protocol was approved by the institutional review boards of Walter Reed Army Medical Center and the National Naval Medical Center in compliance with all applicable federal regulations governing the protection of human subjects. Muscle tissue samples were obtained from two cohorts of subjects after they had provided informed consent. The injured cohort included thirty-one active-duty service members who had sustained blast penetrating extremity injuries (direct exposure to, and open wounds resulting from, high-energy explosives) or non-blast penetrating extremity injuries (high-velocity gunshot injuries). Viable debrided muscle tissue from the wound bed margin was collected at the time of the first wound debridement (four to eight days postinjury) within the continental U.S. Typically, these patients have had two, three, or four previous debridements as they moved through the evacuation process. No study patient received planned prophylaxis against heterotopic ossification with either local irradiation or nonsteroidal anti-inflammatory drugs (NSAIDs). Hamstring (semitendinosus and gracilis) muscle, which served as uninjured control tissue, was collected from six patients undergoing elective anterior cruciate ligament (ACL) reconstruction with hamstring autograft following a non-combat sports-related injury. Criteria for excluding injured patients were concomitant penetrating intracranial injuries, severe thoracic or abdominal injuries, vascular injury requiring repair in the injured limb, and obvious spinal cord injury or upper motor neuron injury. In both cohorts, patients with autoimmune disease, diabetes, a connective-tissue disorder, or any immunocompromised state were also excluded. Patients who had sustained a traumatic brain injury without a penetrating intracranial wound were not excluded.
Muscle Cell Isolation
Muscle tissue, devoid of fascia and fat, was weighed, thoroughly minced into a fine slurry with use of sterile scissors, incubated in 300 U/mL of Collagenase Type II (Worthington, Lakewood, New Jersey) for two hours, rigorously triturated (ten to fifteen times, with use of a 5-mL pipette) to release cells from intact fibers, and then washed to remove cell debris. The cell pellets were resuspended in complete human Mesenchymal Growth Medium (Lonza, Walkersville, Maryland) with antibiotics before serial filtration through sterile 100-μm and then 40-μm nylon filters (Millipore, Billerica, Massachusetts).
Colony-Forming-Unit Assays
Isolated muscle-derived cells were serially diluted and plated into non-coated T25 tissue culture flasks (Fisher Scientific, Pittsburgh, Pennsylvania). Cultures were maintained at 37°C in a humidified atmosphere containing 5% CO2. Cells were allowed to adhere overnight. Non-adherent cells were washed out with medium changes every four days. After eleven to seventeen days of culture, fibroblast-like colonies were counted with use of light phase-contrast microscopy (40× magnification) and/or fixed with methanol, and then they were stained with Giemsa (Sigma-Aldrich, St. Louis, Missouri). Cell clusters consisting of at least thirty fibroblastoid-like cells were considered to be connective-tissue progenitor cells. Photographs were made with use of an inverted BX50 Olympus microscope equipped with an Insight Firewire Spot Color Camera (Diagnostic Instruments, Sterling Heights, Michigan).
Fluorescence-Activated Cell Sorting (FACS) Analysis of Muscle-Derived Connective-Tissue Progenitor Colony-Forming Units
Connective-tissue progenitor colonies were detached with 0.25% trypsin-EDTA (Invitrogen, Rockville, Maryland), harvested, washed twice with staining buffer (Dulbecco phosphate-buffered saline solution plus 5% fetal bovine serum and 0.1% sodium azide), and stained with anti-CD34-fluorescein isothiocyanate (FITC), CD44-FITC, CD45-FITC, HLA-DR-FITC, CD29-phycoerythrin (PE), CD31-PE, CD73-PE, and HLA-ABC-PE (BD Pharingen, San Diego, California). Fluorochrome-labeled irrelevant mouse isotypic mAbs served as controls. At least 104 cells were acquired from each sample and analyzed with use of a BD FACSAria III cell sorter (BD Biosciences, San Jose, California).
Osteogenic Differentiation
Colony-forming-unit cultures were incubated MSC Basal Medium (Lonza) supplemented with osteogenic stimulatory supplements: 10−8 M dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerol phosphate (Lonza) for two to three weeks with complete medium exchanges every four to five days. Mineralized matrix was detected by alizarin red staining. Connective-tissue progenitors that demonstrated in vitro osteogenic potential were classified as connective-tissue progenitor-O, whereas connective-tissue progenitors that gave rise to fibroblastoid colonies with no connective-tissue-differentiation potential were classified as connective-tissue progenitor-F.
Osteogenic Gene Expression
Adherent cells composing primary connective-tissue progenitor colonies were detached by trypsinization, washed, and resuspended in TRIzol (Invitrogen). Total RNA was extracted, and quantitative real-time reverse-transcription polymerase chain reaction assays were performed with use of the human osteogenesis RT2 Profiler polymerase chain reaction array according to the manufacturer's directions (SABiosciences, Gaithersburg, Maryland). Results were analyzed with use of the PCR Array Data Analysis Web Portal (SABiosciences) and normalized to the endogenous housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Comparisons of colony-forming-unit-derived cells were then made between subjects with and those without heterotopic ossification. A gene was reported as differentially regulated if there was a threefold difference in expression.
Assessment of Heterotopic Ossification
The presence of heterotopic ossification was confirmed either by in vivo diagnosis in the operating room during a subsequent surgical debridement or by radiographic follow-up at a minimum of two months following injury. The heterotopic ossification lesions were classified with use of the methodology described by Potter et al.13.
Assessment of Traumatic Brain Injury
Inpatient and outpatient records were reviewed for results of serial traumatic brain injury screening examinations, which all U.S. combat casualties undergo, and findings were compared with the development of heterotopic ossification.
Statistical Analyses
All statistical analyses were performed with use of the Mann-Whitney U test (nonpaired, nonparametric, two-tailed p values; GraphPad PRISM 4.0; GraphPad Software, San Diego, California).
Source of Funding
This work was supported by the Office of Naval Research and U.S. Navy Bureau of Medicine and Surgery under the Medical Development work unit numbers 601152N.00001.2130.A1001 and 604771N.0933.001.A0604.
Subject and Wound Injury Demographics
The control cohort of patients who had undergone harvest of a hamstring tendon autograft consisted of six male patients with an average age of twenty-nine years (range, twenty-three to forty years). The injury cohort consisted of thirty men and one woman with an average age of twenty-four years (range, nineteen to thirty-four years). The injuries were mainly due to a blast mechanism (n = 26), and the remainder were due to gunshot wounds (n = 4) or an explosively formed penetrator (n = 1). In the injured cohort, the muscle biopsy specimens were taken from the sites of nine traumatic amputations, sixteen open fractures, and six deep soft-tissue injuries. All patients underwent initial debridement as a part of definitive surgical care in the U.S. at an average of six days (range, three to eight days) after injury. The results from six patients (one with a traumatic amputation and five with soft-tissue injuries) were excluded from the final comparative results because of inadequate radiographic follow-up to determine whether heterotopic ossification had formed. Two of these patients, with soft-tissue injuries, did not have any postoperative radiographs, and four did not have radiographs after eight days (range, five to eight days) following surgery.
Twenty-five injured patients were thus followed for development of heterotopic ossification after combat-related penetrating trauma (see Appendix). Fourteen demonstrated heterotopic ossification on radiographs obtained at an average of fifty-five days (range, fourteen to 115 days) following injury (Fig. 1). One injury was caused by an explosively formed penetrator, and thirteen were due to improvised explosive devices (IEDs). In the cohort that did not have heterotopic ossification, two injuries were due to gunshot wounds and twelve were due to IEDs. One patient had a mass of heterotopic ossification removed during a surgical debridement twenty-eight days following the initial injury. Within the study period, one patient underwent excision of a symptomatic heterotopic ossification lesion at the distal end of a transtibial amputation on postinjury day 254. Five of the patients with heterotopic ossification had sustained a mild traumatic brain injury due to the combat event. In the group without heterotopic ossification, five patients had sustained a mild traumatic brain injury and one, a moderate traumatic brain injury. None of the remaining patients in either cohort had sustained measurable traumatic brain injury. No patient with a traumatic brain injury developed progressive symptoms as determined with more advanced traumatic brain injury ratings during the course of their treatment and recovery.
Muscle-Derived Fibroblast Colony-Forming Progenitor Cells
A small percentage (0.05% to 3%) of the nucleated cells derived from the digested muscle tissue were plastic adherent and remained quiescent for three to five days before they started dividing to form proliferative foci of adherent cells. After seven to ten days, single cells gave rise to individual stromal-cell-like fibroblast colony-forming units that were visualized and counted with use of phase-contrast light microscopy or Giemsa staining (Fig. 2, A). Colony-forming-unit colonies were heterogeneous in size and cell density, suggesting differences in the rate of progenitor cell proliferation. As compared with the cells from the control group, adherent cells from the injured cohort developed sooner, expanded more rapidly, and formed a subconfluent adherent monolayer consisting of distinct individual colonies sooner (average, thirteen days [range, ten to seventeen days] compared with nineteen days [range, seventeen to twenty days] in the normal group).
Expression of Cell Surface Antigens on Progeny Cells of Connective-Tissue Progenitors
We used flow cytometric analysis to examine the presence of surface antigens (Table I) on the progeny of connective-tissue progenitors (passage 0). Cells were found to be strongly positive for CD29 (β-integrin), CD44 (hyaluronate receptor), CD73 (ecto-5-prime-nucleotidase), and HLA-ABC (MHC-1). In contrast, cells were negative for lineage-positive cells (CD4, CD8, CD11b, CD14, and CD19; data not shown), the leukocyte marker CD45, the hematopoietic stem cell marker CD34, the endothelial cell marker CD31 (platelet/endothelial cell adhesion molecule [PECAM-1]), and the major histocompatibility complex (MHC) class-II-HLA-DR antigen complex (Fig. 2, B).
Multipotent Differentiation Potential of Connective-Tissue Progenitor Progeny Cells
A small percentage of the muscle-derived colony-forming units from normal muscle tissue had the capacity to differentiate into diverse mesodermal cell types such as osteoblasts, chondrocytes, and adipocytes. A larger percentage of the connective-tissue progenitors demonstrated no connective-tissue differentiation potential and thus were considered to have been derived from connective-tissue fibroblast colony-forming progenitor cells (connective-tissue progenitor-F) (data not shown). No mineralized matrix was observed in cells maintained under basal growth medium conditions.
Assessment of Connective-Tissue Progenitor Content in Injured Tissue
In the control cohort (n = 6), there were an average (and standard error of the mean [SEM]) of 4341 ± 879 connective-tissue progenitor colonies per gram of tissue (range, 1077 to 7594). Injured patients (n = 25) had significantly higher numbers of muscle-derived connective-tissue progenitor cells per gram of tissue (average, 196,023 ± 14,600; p < 0.0001; 95% confidence interval [CI], 129,930 to 253,333). Within the injured cohort, the specimens from the wounds in which heterotopic ossification did not develop (n = 11) yielded an average of 164,636 ± 14,755 connective-tissue progenitor colonies per gram of tissue, and those from the wounds in which heterotopic ossification did develop (n = 14) yielded significantly more connective-tissue progenitors per gram of tissue (average, 220,684 ± 20,774; p < 0.048; 95% CI, 409 to 111,687) (Fig. 3-A).
Osteogenic Differentiation of Muscle-Derived Colony-Forming Units
The mean number of connective-tissue progenitor-O colonies per gram of tissue in the injured patients who subsequently developed heterotopic ossification (163,991 ± 18,292; range, 33,600 to 307,273; n = 14) was significantly higher (p < 0.016; 95% CI, 12,249 to 106,065) than that in the injured patients who had healing without heterotopic ossification formation (104,834 ± 10,424; range, 61,600 to 181,818; n = 11) (Fig. 3-A). Moreover, the percentage of connective-tissue progenitors demonstrating osteogenic potential was greater in the patients with heterotopic ossification than in those without heterotopic ossification (74% and 64%, respectively), although the detected difference was not significant (Fig. 3-B).
No significant differences in the numbers of all connective-tissue progenitor colonies, connective-tissue progenitor-O colonies, or connective-tissue progenitor-F colonies (data not shown) were detected between the muscle tissues collected from the patient with mild heterotopic ossification and the muscle tissues collected from the patients who developed moderate-to-severe heterotopic ossification.
Osteogenic Gene Expression in Connective-Tissue Progenitor Progeny Cells
Quantitative real-time reverse-transcription polymerase chain reaction was performed with use of connective-tissue progenitor progeny cells (passage 0) to evaluate the early differential expression of osteogenic-related genes in injured and healing muscle tissue (Fig. 4). As compared with connective-tissue progenitor-derived progeny cells from tissues collected from patients in whom the wound had healed without heterotopic ossification formation (n = 7), connective-tissue progenitor-derived progeny cells from patients who subsequently developed heterotopic ossification in the wound (n = 12) expressed higher amounts of collagen type-X alpha (COL10A1); collagen-4-alpha-3 (COL4A3) (p < 0.05; 95% CI; 0.36 to 4.38); cartilage oligomeric matrix protein (COMP); fibroblast growth factor receptor-2 (FGFR2); vascular endothelial growth factor receptor-1 (FLT1); insulin-like growth factor 2 (IGF2); integrin, alpha-M (ITGAM); matrix metallopeptidase-9/gelatinase (MMP9); phosphate-regulating neutral endopeptidase (PHEX); scavenger receptor class B, member 1 (SCARB1); transcription factor SOX9; and vascular endothelial growth factor-A (VEGFA). In this analysis, we observed that seven gene transcripts associated with growth factors/receptors and transcriptional regulation were modestly, but not significantly (p > 0.05), down-regulated compared with those in the injured group without heterotopic ossification.
The effects of traumatic injury on resident osteogenic progenitor cell populations endogenous to and/or mobilized to the wound microenvironment have not been previously characterized either quantitatively or qualitatively, to our knowledge. In this study, we demonstrated that wounds that present with a higher prevalence of assayable osteoprogenitors in the tissue correlate with the eventual formation of ectopic bone in the traumatized tissue. We believe this to be the first study in which the connective-tissue progenitor, connective-tissue progenitor-O, and connective-tissue progenitor-F muscle-derived colony-forming cell content in the wound margin/granulation tissue from patients who developed heterotopic ossification was quantitatively and qualitatively assessed and compared with that in similarly injured patients who had healing without complications. Results from this study provide further biological insight into the heterotopic ossification pathological process.
In this study, as early as ninety-six hours postinjury, elevated numbers of connective-tissue progenitors could be detected in various types of acute traumatic combat wounds, including soft-tissue gunshot wounds, complex open tibial and femoral fractures, and transtibial and transfemoral amputations. The prevalences of connective-tissue progenitor and connective-tissue progenitor-O colonies in these wounds were found to be thirty-eight to fifty-one-fold and 106 to 166-fold greater, respectively, relative to those detected in uninjured normal hamstring muscle. Post hoc analysis revealed that the 1.56-fold increase in the average number of osteogenic colony-forming (connective-tissue progenitor-O) colonies per gram of tissue in the injured patients who developed heterotopic ossification, as compared with the average number in those who did not, was significant (p < 0.016; 95% CI, 12,249 to 106,065). There was also a significant, 1.34-fold increase in the average number of connective-tissue progenitor colonies per gram of tissue in the injured patients who developed heterotopic ossification compared with the average number in those who did not (p < 0.048; 95% CI, 409 to 111,687). Many of the connective-tissue progenitors, especially those isolated from normal tissue (∼75%), failed to differentiate into any of the mesenchymal lineages tested (bone, cartilage, or fat) and thus were considered to be fibroblasts (connective-tissue progenitor-F). Progeny cells composing primary muscle-derived connective-tissue progenitor colony-forming units were neither hematopoietic cells (i.e., were negative for CD45 and HLA-DR) nor endothelial cells (were negative for CD34 and CD31) and were positive for CD29, CD44, CD73, and HLA-ABC. Moreover, we observed that early progeny cells from these progenitor cells developed more quickly, developed into larger colonies, had a transcriptional expression signature characteristic of early osteogenic differentiation, and demonstrated a greater propensity in culture to differentiate along the osteogenic lineage, suggesting that there may be early correlation with the onset and/or progression of heterotopic ossification.
The colony-forming-assay system used in this study must be interpreted as a means of estimating the concentration and prevalence of tissue progenitors. The observed prevalence may be an underestimate of the true prevalence. Some progenitors in the tissue may not have been detected because of either loss in the initial processing of tissue or limitations in the colony-forming efficiency in the assay (i.e., the probability that a progenitor in a given sample will form a colony that is counted will likely be less than 1.0). Further analysis is under way to more precisely determine and optimize colony-forming efficiency. Further work is also ongoing to more rigorously characterize cell phenotypes and the capacity of the progeny of individual colonies for multilineage differentiation in an attempt to define parameters with greater predictive value for the formation of clinical heterotopic ossification.
A critical translational aspect of this study is its prospective, "blinded" design. Tissue samples were obtained and analyzed weeks to months before radiographically evident heterotopic ossification was detected, and our in vitro assessment was performed at the single-progenitor-cell level (four, five, or six cell divisions with seven to ten days of culture). At this time, we cannot definitively either rule out or rule in the participation of unipotent and/or multipotent colony-forming progenitor cells with reparative functions that may have been recruited and/or mobilized remotely after injury from other regions, such as the bone marrow. Consistent with our observations, Nesti et al.16 and Jackson et al.17 showed that multipotent progenitor cells with osteogenic potential are present in combat-injured muscle tissue. In our studies, we examined the cell progeny of connective-tissue progenitor colonies prior to the first passage to avoid the alteration of osteogenic expression based on intrinsic cell changes linked with prolonged culture expansion conditions.
Bone regeneration is characterized by extensive matrix remodeling involving cartilage formation, mineralization, and replacement by immature woven bone, which is ultimately remodeled to lamellar bone. These processes have been shown to be regulated both spatially and temporally22. It is thought that resident tissue musculoskeletal-specific mesenchymal stem cells/connective-tissue progenitors play a key role in bone regeneration, possibly acting via migration into the adjacent site of injury, contributing biological factors important in healing, and then proliferating and differentiating into functional cells of the mesenchymal lineages23. Our findings indicate that, early in the wound repair response, the progeny of the connective-tissue progenitors derived from patients who developed heterotopic ossification showed higher expression of COL10A1, COL4A3, COMP, FGFR2, FLT1, IGF2, ITGAM, MMP9, PHEX, SCARB1, SOX9, and VEGFA. Regarding the transforming growth factor/bone morphogenetic protein (TGF/BMP) superfamily, no increases in BMP2 and BMP4 expression were noted. Furthermore, we detected no significant change in Runx2 transcription and its downstream targets related to bone matrix extracellular proteins (ALP, BGN, osteocalcin/BGP, BSP, and OSX)24. These findings support our observations that osseous matrix was not detectable in non-induced cells cultures by staining with alizarin red. These findings are not surprising as bone extracellular matrix expression is barely detectable in proliferative and non-induced cells25-27.
After induction of differentiation, however, osteogenic gene expression increases dramatically in parallel with mineral deposition25,28. In contrast, a measured increase in SOX9, a potent osteogenic transcription factor that commits osteogenic stem/progenitor cells to the skeletal lineage, was observed29. Secretion of COL10A1, and COMP, a major noncollagenous component of cartilage extracellular matrix, localized in adult and fetal osteoblasts has been shown to play an important role in chondrogenesis and endochondral bone growth30. The cartilage-degradable proteinases MMP9 and MMP13 are found in bone-forming osteoblasts and play critical roles in vascular invasion31,32. In mesenchymal stem cell-derived osteoprogenitor cells, heightened MMP9 synthesis and cartilage vascularization has been reported following proinflammatory cytokine (tumor necrosis factor-alpha [TNF-alpha], interleukin-1-alpha [IL-1 alpha], and TGF-beta) signaling33. VEGFA and the corresponding receptors VEGFR-1 (FLT1) and VEGFR-2 (FLK-1) play distinct roles in osteoblast differentiation, angiogenic vascularization, and mineralized bone formation34. Similarly, FGFs and IGFs regulate bone formation as endothelial mitogens, whereas FGF-1 supports immature osteogenic progenitor cell proliferation35. Although the exact interactions of these expressed transcripts in primary muscle-derived connective-tissue progenitors have not been elucidated, the upregulation of a number of key genes involving matrix synthesis, vascularization, and early mineralization phases of endochondral ossification correlates with the observed heightened osteogenic differentiation capacity of connective-tissue progenitor to connective-tissue progenitor-O under osteogenic inductive conditions. These findings suggest that the progenitor population in these wounds at the time of the first debridement is committed to a connective-tissue phenotype but is not yet committed to a bone phenotype. If this is true, this finding opens the opportunity to define assay criteria to detect patients who are at high risk for heterotopic ossification and intervene in a manner that inhibits bone differentiation as an outcome.
Although we attempted to control for confounding injuries and preexisting medical conditions, several factors related to the treatment of wartime injuries may have contributed to the eventual development of heterotopic ossification. A majority of open wounds in this setting are managed with negative-pressure wound therapy at some point in the treatment course. The constant mechanical stress on the wound while the device is in place may create a shearing force on the muscle fibers, perpetuating a local insult to the tissues that is essential to the formation of heterotopic ossification36. Additionally, Knippenberg et al.37 found that adipose tissue-derived mesenchymal stem cells show a bone-cell-like response to pulsating fluid shear stress.
The present study provides important insight into the mechanisms of heterotopic ossification formation. This methodology might prove useful to not only assess whether heterotopic ossification will ultimately develop in a wound, but also to guide future early prophylactic measures tailored to the individual patient to prevent formation of heterotopic ossification.
A table showing patient demographics, information regarding the wounds, and the radiographic grades of the heterotopic ossification is available with the online version of this article at jbjs.org.
Note: The authors thank Stephen Zins for technical laboratory support, Doug Smoot for assistance with flow cytometry, Fred Gage for administrative assistance, and Dr. Douglas Tadaki (Naval Medical Research Center) and Dr. Jonathan Forsberg (National Naval Medical Center) for helpful discussions in the early stages of this project. They thank members of both the clinical and the research laboratories for stimulating discussions and support throughout the course of these studies.
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