Early local delivery of vancomycin suppresses ectopic bone formation in a rat model of trauma-induced heterotopic ossification.

Heterotopic ossification (HO) is a debilitating sequela of high-energy injuries. It frequently requires surgical excision once symptomatic and there is no practical prophylaxis for combat-injured patients. In this study, we examined the effect of local vancomycin powder on HO formation in a small animal model of blast-related, post-traumatic HO. Male Sprague-Dawley rats were subjected to a polytraumatic extremity injury and amputation with or without methicillin-resistant Staphylococcus aureus infection. Animals were randomized to receive a single local application of vancomycin (20 mg/kg) at the time of injury (POD-0, n = 34) or on postoperative day-3 (POD-3, n = 11). Quantitative volumetric measurement of ectopic bone was calculated at 12-weeks post-injury by micro-CT. Bone marrow and muscle tissues were also collected to determine the bacterial burden. Blood for serum cytokine analysis was collected at baseline and post-injury. Vancomycin treatment on POD-0 suppressed HO formation by 86% and prevented bone marrow and soft tissue infections. We concurrently observed a marked reduction histologically in nonviable tissue, chronic inflammatory cell infiltrates, bone infection, fibrous tissue, and areas of bone necrosis within this same cohort. Delayed treatment was significantly less efficacious. Neither treatment had a marked effect on the production of pro-inflammatory cytokines. Our study demonstrates that local vancomycin treatment at the time of injury significantly reduces HO formation in both the presence and absence of infection, with decreased efficacy if not given early. These findings further support the concept that the therapeutic window for prophylaxis is narrow, highlighting the need to develop early treatment strategies for clinical management. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2397-2406, 2017.

Scleraxis-Lineage Cells Contribute to Ectopic Bone Formation in Muscle and Tendon.

The pathologic development of heterotopic ossification (HO) is well described in patients with extensive trauma or with hyperactivating mutations of the bone morphogenetic protein (BMP) receptor ACVR1. However, identification of progenitor cells contributing to this process remains elusive. Here we show that connective tissue cells contribute to a substantial amount of HO anlagen caused by trauma using postnatal, tamoxifen-inducible, scleraxis-lineage restricted reporter mice (Scx-creERT2/tdTomatofl/fl ). When the scleraxis-lineage is restricted specifically to adults prior to injury marked cells contribute to each stage of the developing HO anlagen and coexpress markers of endochondral ossification (Osterix, SOX9). Furthermore, these adult preinjury restricted cells coexpressed mesenchymal stem cell markers including PDGFRα, Sca1, and S100A4 in HO. When constitutively active ACVR1 (caACVR1) was expressed in scx-cre cells in the absence of injury (Scx-cre/caACVR1fl/fl ), tendons and joints formed HO. Postnatal lineage-restricted, tamoxifen-inducible caACVR1 expression (Scx-creERT2/caACVR1fl/fl ) was sufficient to form HO after directed cardiotoxin-induced muscle injury. These findings suggest that cells expressing scleraxis within muscle or tendon contribute to HO in the setting of both trauma or hyperactive BMP receptor (e.g., caACVR1) activity. Stem Cells 2017;35:705-710.

Characterization of Cells Isolated from Genetic and Trauma-Induced Heterotopic Ossification.

Heterotopic ossification (HO) is the pathologic formation of bone separate from the normal skeleton. Although several models exist for studying HO, an understanding of the common in vitro properties of cells isolated from these models is lacking. We studied three separate animal models of HO including two models of trauma-induced HO and one model of genetic HO, and human HO specimens, to characterize the properties of cells derived from tissue containing pre-and mature ectopic bone in relation to analogous mesenchymal cell populations or osteoblasts obtained from normal muscle tissue. We found that when cultured in vitro, cells isolated from the trauma sites in two distinct models exhibited increased osteogenic differentiation when compared to cells isolated from uninjured controls. Furthermore, osteoblasts isolated from heterotopic bone in a genetic model of HO also exhibited increased osteogenic differentiation when compared with normal osteoblasts. Finally, osteoblasts derived from mature heterotopic bone obtained from human patients exhibited increased osteogenic differentiation when compared with normal bone from the same patients. These findings demonstrate that across models, cells derived from tissues forming heterotopic ossification exhibit increased osteogenic differentiation when compared with either normal tissues or osteoblasts. These cell types can be used in the future for in vitro investigations for drug screening purposes.

Bioburden Increases Heterotopic Ossification Formation in an Established Rat Model.

BACKGROUND: Heterotopic ossification (HO) develops in a majority of combat-related amputations wherein early bacterial colonization has been considered a potential early risk factor. Our group has recently developed a small animal model of trauma-induced HO that incorporates many of the multifaceted injury patterns of combat trauma in the absence of bacterial contamination and subsequent wound colonization. QUESTIONS/PURPOSES: We sought to determine if (1) the presence of bioburden (Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus [MRSA]) increases the magnitude of ectopic bone formation in traumatized muscle after amputation; and (2) what persistent effects bacterial contamination has on late microbial flora within the amputation site. METHODS: Using a blast-related HO model, we exposed 48 rats to blast overpressure, femur fracture, crush injury, and subsequent immediate transfemoral amputation through the zone of injury. Control injured rats (n = 8) were inoculated beneath the myodesis with phosphate-buffered saline not containing bacteria (vehicle) and treatment rats were inoculated with 1 × 10(6) colony-forming units of A baumannii (n = 20) or MRSA (n = 20). All animals formed HO. Heterotopic ossification was determined by quantitative volumetric measurements of ectopic bone at 12-weeks postinjury using micro-CT and qualitative histomorphometry for assessment of new bone formation in the residual limb. Bone marrow and muscle tissue biopsies were collected from the residual limb at 12 weeks to quantitatively measure the bioburden load and to qualitatively determine the species-level identification of the bacterial flora. RESULTS: At 12 weeks, we observed a greater volume of HO in rats infected with MRSA (68.9 ± 8.6 mm(3); 95% confidence interval [CI], 50.52-85.55) when compared with A baumannii (20.9 ± 3.7 mm(3); 95% CI, 13.61-28.14; p < 0.001) or vehicle (16.3 ± 3.2 mm(3); 95% CI, 10.06-22.47; p < 0.001). Soft tissue and marrow from the residual limb of rats inoculated with A baumannii tested negative for A baumannii infection but were positive for other strains of bacteria (1.33 × 10(2) ± 0.89 × 10(2); 95% CI, -0.42 × 10(2)-3.08 × 10(2) and 1.25 × 10(6) ± 0.69 × 10(6); 95% CI, -0.13 × 10(6)-2.60 × 10(6) colony-forming units in bone marrow and muscle tissue, respectively), whereas tissue from MRSA-infected rats contained MRSA only (4.84 × 10(1) ± 3.22 × 10(1); 95% CI, -1.47 × 10(1)-11.1 × 10(1) and 2.80 × 10(7) ± 1.73 × 10(7); 95% CI, -0.60 × 10(7)-6.20 × 10(7) in bone marrow and muscle tissue, respectively). CONCLUSIONS: Our findings demonstrate that persistent infection with MRSA results in a greater volume of ectopic bone formation, which may be the result of chronic soft tissue inflammation, and that early wound colonization may be a key risk factor. CLINICAL RELEVANCE: Interventions that mitigate wound contamination and inflammation (such as early débridement, systemic and local antibiotics) may also have a beneficial effect with regard to the mitigation of HO formation and should be evaluated with that potential in mind in future preclinical studies.

Electrospun bio-nanocomposite scaffolds for bone tissue engineering by cellulose nanocrystals reinforcing maleic anhydride grafted PLA.

Electrospun fibrous bio-nanocomposite scaffolds reinforced with cellulose nanocrystals (CNCs) were fabricated by using maleic anhydride (MAH) grafted poly(lactic acid) (PLA) as matrix with improved interfacial adhesion between the two components. Morphological, thermal, mechanical, and in vitro degradation properties as well as basic cytocompatibility using human adult adipose derived mesenchymal stem cells (hASCs) of MAH grafted PLA/CNC (i.e., MPLA/CNC) scaffolds were characterized. Morphological investigation indicated that the diameter and polydispersity of electrospun MPLA/CNC nanofibers were reduced with the increased CNC content. The addition of CNCs improved both the thermal stability and mechanical properties of MPLA/CNC composites. The MPLA/CNC scaffolds at the 5 wt % CNC loading level showed not only superior tensile strength (more than 10 MPa), but also improved stability during in vitro degradation compared with the MPLA and PLA/CNC counterparts. Moreover, the fibrous MPLA/CNC composite scaffolds were non-toxic to hASCs and capable of supporting cell proliferation. This study demonstrates that fibrous MPLA/CNC bio-nanocomposite scaffolds are biodegradable, cytocompatible, and possess useful mechanical properties for bone tissue engineering.