Jonathan A. Forsberg

Metalloproteinase Expression is Associated with Traumatic Wound Failure

BACKGROUND Matrix metalloproteinases (MMPs) are crucial in the inflammatory and remodeling phases of wound healing. We previously reported the correlation between pro-inflammatory cytokines and timing of successful combat-wound closure. We now extend our studies to investigate the correlation between wound-remodeling MMP expression and wound healing. METHODS Thirty-eight wounds in 25 patients with traumatic extremity combat wounds were prospectively studied. Surgical debridement with vacuum-assisted closure (VAC) device application was repeated every 48 to 72h until surgical wound closure. Wound effluent and patient serum were collected at each wound debridement and analyzed for five matrix metalloproteinases using the Luminex multiplex system; Millipore Corp, Billerica, MA. The primary outcome was wound healing within 30 d of definitive wound closure. Impairment was defined as delayed wound closure (>21 d from injury) or wound dehiscence. MMP expression was compared between impaired and normal healing wounds. RESULTS Elevated levels of serum MMP-2 and MMP-7 and reduced levels of effluent MMP3 were seen in impaired wounds (n = 9) compared with wounds that healed (n = 29; P<0.001). Receiver operating characteristic (ROC) curve analysis yielded area-under-the-curve (AUC) of 0.744, 0.783, and 0.805, respectively. CONCLUSIONS Impaired wound healing is characterized by pro-inflammatory MMP-2 and MMP-7. Serum and effluent concentrations of MMP-2, MMP-3, and MMP-7 can effectively predict the outcome of traumatic war wounds and can potentially provide decision-supportive, objective evidence for the timing of wound closure.

Chronic Lateral Ankle Instability and Associated Conditions: A Rationale for Treatment:

Background: Ankle sprains have a high incidence of associated injuries and conditions that may be unrecognized at the initial time of injury. Failure to treat these conditions at the index surgery may compromise outcomes and delay recovery. The purpose of this study was to determine the type and frequency of associated injuries and conditions in military patients with chronic lateral ankle instability. Methods: Between 1996 and 2002, 160 patients had 180 modified Broström-Gould lateral ankle ligament reconstructions for chronic ankle instability. A retrospective review of the clinical history, physical examination, radiographs, and intraoperative findings was conducted. Results: The overall incidence of associated extra-articular conditions and injuries found in this study was 64%; 115 conditions were identified in 180 ankles. Peroneal tendon injuries occurred with the highest frequency (28%), followed by os trigonum lesions (13%), lateral gutter ossicles (10%), hindfoot varus alignment (8%), anterior tibial spurs (3%), and tarsal coalitions (2%). Twenty revision lateral ankle ligament reconstructions were required for either persistent pain or recurrent instability. The most common associated conditions were undiagnosed hindfoot varus alignment abnormalities (28%) followed by untreated peroneal injuries (25%). Conclusions: This study confirms the frequency of conditions associated with lateral ankle instability and emphasizes several conditions that have received little attention in the literature. Identifying these associated conditions before surgery enables the surgeon to treat all conditions at one operation, returning the patient to full activity sooner. Guidelines are presented to assist clinicians in screening patients for these associated conditions.

Diagnosis and Management of Chronic Infection

&NA; High‐energy penetrating extremity injuries are often associated with severe open fractures that have varying degrees of soft‐tissue contamination and tenuous soft‐tissue coverage. The result is a relatively high prevalence of chronic osteomyelitis compared with that in civilian trauma patients. Diagnosing chronic osteomyelitis requires a careful history and thorough physical and radiographic examinations. Cross‐sectional imaging can help delineate the extent of bony involvement, and scintigraphy can be used as a diagnostic tool and to gauge response to treatment. Clinical staging also directs surgical management. Adequacy of débridement remains the most important clinical predictor of success; thus, adopting an oncologic approach to complete (ie, wide) excision is important. Reconstruction can be safely performed by a variety of methods; however, proper staging and patient selection remain critical to a successful outcome. Although systemic and depot delivery of antibiotics plays a supporting role in the treatment of chronic osteomyelitis, the ideal dosing regimens, and the duration of treatment, remain controversial.

Raman spectroscopic analysis of combat-related heterotopic ossification development☆

Over 60% of our severely combat-injured patient population develops radiographically apparent heterotopic ossification. Nearly a third of these require surgical excision of symptomatic lesions, a procedure that is fraught with complications, and delays or regresses functional rehabilitation in many cases. Unfortunately, for the combat injured, medical contraindications and logistical limitations limit widespread use of conventional means of primary prophylaxis. Better means of risk stratification are needed to both mitigate the risk of current means of primary prophylaxis as well as to evaluate novel preventive strategies currently in development. We asked whether Raman spectral changes, measured ex vivo, correlated with histologic evidence of the earliest signs of HO formation using tissue biopsies from the wounds of combat casualties. In doing so, we compared normal muscle tissue to injured muscle tissue, unmineralized HO tissue, and mineralized HO tissue. The Raman spectra of these tissues demonstrate clear differences in the amide I and amide III spectral regions of HO tissue compared to normal tissue, denoted by changes in the 1640/1445cm(-1)(p<0.01), and 1340/1270cm(-1) (p<0.01) band area ratios (BARs). Additionally, analysis of the bone mineral in HO by Raman spectroscopy appears capable of determining bone maturity by measuring both the 945/960cm(-1) and the 1070/1445cm(-1) BARs. Raman may therefore prove a useful, non-invasive, and early diagnostic modality to detect HO formation prior to it becoming evident clinically or radiographically. This technique could ostensibly be utilized as a non-invasive means to risk stratify individual wounds at a time thought to be amenable to various means of primary prophylaxis.

Microbial profiling of combat wound infection through detection microarray and next-generation sequencing

ABSTRACT Combat wound healing and resolution are highly affected by the resident microbial flora. We therefore sought to achieve comprehensive detection of microbial populations in wounds using novel genomic technologies and bioinformatics analyses. We employed a microarray capable of detecting all sequenced pathogens for interrogation of 124 wound samples from extremity injuries in combat-injured U.S. service members. A subset of samples was also processed via next-generation sequencing and metagenomic analysis. Array analysis detected microbial targets in 51% of all wound samples, with Acinetobacter baumannii being the most frequently detected species. Multiple Pseudomonas species were also detected in tissue biopsy specimens. Detection of the Acinetobacter plasmid pRAY correlated significantly with wound failure, while detection of enteric-associated bacteria was associated significantly with successful healing. Whole-genome sequencing revealed broad microbial biodiversity between samples. The total wound bioburden did not associate significantly with wound outcome, although temporal shifts were observed over the course of treatment. Given that standard microbiological methods do not detect the full range of microbes in each wound, these data emphasize the importance of supplementation with molecular techniques for thorough characterization of wound-associated microbes. Future application of genomic protocols for assessing microbial content could allow application of specialized care through early and rapid identification and management of critical patterns in wound bioburden.

Ectopic bone formation in severely combat-injured orthopedic patients -- a hematopoietic niche.

Combat-related heterotopic ossification (HO) has emerged as a common and problematic complication of modern wartime extremity injuries, contributing to substantial patient morbidity and loss of function. We have previously reported that HO-forming patients exhibit a more pronounced systemic and local inflammatory response very early in the wound healing process. Moreover, traumatized muscle-derived mesenchymal progenitor cells from these patients have a skewed differentiation potential toward bone. Here, we demonstrate that HO lesions excised from this patient population contain highly vascularized, mature, cancellous bone containing adipogenic marrow. Histologic analysis showed immature hematopoietic cells located within distinct foci in perivascular regions. The adipogenic marrow often contained low numbers of functional erythroid (BFU-E), myeloid (CFU-GM, CFU-M) and multilineage (CFU-GEMM) colony-forming hematopoietic progenitor cells (HPCs). Conversely, tissue from control muscle and non-HO traumatic wound granulation tissue showed no evidence of hematopoietic progenitor cell activity. In summary, our findings suggest that ectopic bone can provide an appropriate hematopoietic microenvironment for supporting the proliferation and differentiation of HPCs. This reactive and vibrant cell population may help maintain normal hematopoietic function, particularly in those with major extremity amputations who have sustained both massive blood loss, prompting systemic marrow stimulation, as well as loss of available native active marrow space. These findings begin to characterize the functional biology of ectopic bone and elucidate the interactions between HPC and non-hematopoietic cell types within the ectopic intramedullary hematopoietic microenvironmental niche identified.

Impact of Margin Status and Local Recurrence on Soft-tissue Sarcoma Outcomes

BACKGROUND The impact of local recurrence and surgical resection margin status on survival in extremity soft-tissue sarcomas remains to be clearly defined. Our aim was to conduct a retrospective analysis of prospectively collected data to determine the prognostic relevance of positive resection margins and local recurrence for extremity soft-tissue sarcomas for survival. METHODS Three hundred and sixty-three patients who underwent resection of localized primary extremity soft-tissue sarcomas with curative intent were selected from the United States Department of Defense Automated Central Tumor Registry. Outcomes for local recurrence, distant recurrence, disease-specific survival, and overall survival were analyzed according to clinical, pathological, and treatment variables with use of the Kaplan-Meier method (log-rank test) and the multivariate Cox regression model. RESULTS Positive margins (hazard ratio, 1.99 [95% confidence interval, 1.15 to 3.45]), local recurrence (hazard ratio, 2.93 [95% confidence interval, 1.38 to 6.23]), and distant recurrence (hazard ratio, 12.13 [95% confidence interval, 5.97 to 24.65]) were significantly associated with overall survival on multivariate Cox regression analysis. However, for disease-specific survival, local recurrence was not significant and tumor size of >10 cm (hazard ratio, 2.83 [95% confidence interval, 1.15 to 6.95]), positive margins (hazard ratio, 1.95 [95% confidence interval, 1.05 to 3.63]), and distant recurrence (hazard ratio, 9.46 [95% confidence interval, 4.37 to 20.47]) were independent adverse prognostic factors. The disease-specific survival rate for patients with localized soft-tissue sarcomas was 89% (95% confidence interval, 85% to 92%) for five years and 75% (95% confidence interval, 70% to 81%) for ten years. CONCLUSIONS Positive surgical margins are consistently associated with adverse survival-related outcomes in localized soft-tissue sarcomas of the extremity. Local recurrence had a significant impact on overall survival, but not on disease-specific survival.

Targeted stimulation of retinoic acid receptor-γ mitigates the formation of heterotopic ossification in an established blast-related traumatic injury model

Heterotopic ossification (HO) involves formation of endochondral bone at non-skeletal sites, is prevalent in severely wounded service members, and causes significant complications and delayed rehabilitation. As common prophylactic treatments such as anti-inflammatory drugs and irradiation cannot be used after multi-system combat trauma, there is an urgent need for new remedies. Previously, we showed that the retinoic acid receptor γ agonist Palovarotene inhibited subcutaneous and intramuscular HO in mice, but those models do not mimic complex combat injury. Thus, we tested Palovarotene in our validated rat trauma-induced HO model that involves blast-related limb injury, femoral fracture, quadriceps crush injury, amputation and infection with methicillin-resistant Staphylococcus aureus from combat wound infections. Palovarotene was given orally for 14days at 1mg/kg/day starting on post-operative day (POD) 1 or POD-5, and HO amount, wound dehiscence and related processes were monitored for up to 84days post injury. Compared to vehicle-control animals, Palovarotene significantly decreased HO by 50 to 60% regardless of when the treatment started and if infection was present. Histological analyses showed that Palovarotene reduced ectopic chondrogenesis, osteogenesis and angiogenesis forming at the injury site over time, while fibrotic tissue was often present in place of ectopic bone. Custom gene array data verified that while expression of key chondrogenic and osteogenic genes was decreased within soft tissues of residual limb in Palovarotene-treated rats, expression of cartilage catabolic genes was increased, including matrix metalloproteinase-9. Importantly, Palovarotene seemed to exert moderate inhibitory effects on wound healing, raising potential safety concerns related to dosing and timing. Our data show for the first time that Palovarotene significantly inhibits HO triggered by blast injury and associated complications, strongly indicating that it may prevent HO in patients at high risk such as those sustaining combat injuries and other forms of blast trauma.

Wound outcome in combat injuries is associated with a unique set of protein biomarkers

BackgroundThe ability to forecast whether a wound will heal after closure without further debridement(s), would provide substantial benefits to patients with severe extremity trauma.MethodsWound effluent is a readily available material which can be collected without disturbing healthy tissue. For analysis of potential host response biomarkers, forty four serial combat wound effluent samples from 19 patients with either healing or failing traumatic- and other combat-related wounds were examined by 2-D DIGE. Spot map patterns were correlated to eventual wound outcome (healed or wound failure) and analyzed using DeCyder 7.0 and differential proteins identified via LC-MS/MS.ResultsThis approach identified 52 protein spots that were differentially expressed and thus represent candidate biomarkers for this clinical application. Many of these proteins are intimately involved in inflammatory and immune responses. Furthermore, discriminate analysis further refined the 52 differential protein spots to a smaller subset of which successfully differentiate between wounds that will heal and those that will fail and require further surgical intervention with greater than 83% accuracy.ConclusionThese results suggest candidates for a panel of protein biomarkers that may aid traumatic wound care prognosis and treatment. We recommend that this strategy be refined, and then externally validated, in future studies of traumatic wounds.

Lessons of War: Turning Data Into Decisions

Background Recent conflicts in Afghanistan and Iraq produced a substantial number of critically wounded service-members. We collected biomarker and clinical information from 73 patients who sustained 116 life-threatening combat wounds, and sought to determine if the data could be used to predict the likelihood of wound failure. Methods From each patient, we collected clinical information, serum, wound effluent, and tissue prior to and at each surgical débridement. Inflammatory cytokines were quantified in both the serum and effluent, as were gene expression targets. The primary outcome was successful wound healing. Computer intensive methods were used to derive prognostic models that were internally validated using target shuffling and cross-validation methods. A second cohort of eighteen critically injured civilian patients was evaluated to determine if similar inflammatory responses were observed. Findings The best-performing models enhanced clinical observation with biomarker data from the serum and wound effluent, an indicator that systemic inflammatory conditions contribute to local wound failure. A Random Forest model containing ten variables demonstrated the highest accuracy (AUC 0.79). Decision Curve Analysis indicated that the use of this model would improve clinical outcomes and reduce unnecessary surgical procedures. Civilian trauma patients demonstrated similar inflammatory responses and an equivalent wound failure rate, indicating that the model may be generalizable to civilian settings. Interpretation Using advanced analytics, we successfully codified clinical and biomarker data from combat patients into a potentially generalizable decision support tool. Analysis of inflammatory data from critically ill patients with acute injury may inform decision-making to improve clinical outcomes and reduce healthcare costs. Funding United States Department of Defense Health Programs.