Experimental Induction of Physeal Injuries by Fracture, Drill, and Ablation Techniques: Analyses of Immunohistochemical Findings.

BACKGROUND: Although physeal fractures and physeal bars can result in significant clinical consequences to growth and development of the injured physis, little orthopaedic research has focused upon this topic. Our objective was to extend a previously developed rat model to examine the immunohistochemical features following surgical application of techniques disrupting the physis. METHODS: Physes were surgically disrupted using fracture (control), epiphyseal scrape (ES), or epiphyseal drill (ED). After 1, 3, 6, 10, or 21 days, animals were euthanized, sites processed for histology and immunohistochemical localization of vascular endothelial growth factor (VEGF), Factor VIII, Sox-9, PTHrP (parathyroid hormone-related protein) and PTHrP-R (parathyroid hormone-related protein receptor) in resting, proliferative, and hypertrophic physeal zones. Incidence of physeal bars, vertical septa and islands within the metaphysis was quantified. Semiquantitative analysis of immunohistochemistry was performed. RESULTS: Physeal bars, vertical septa, and displaced cartilage islands were present each of the surgical treatments. Fisher's exact test showed a statistically significant increase in the presence of physeal bars (P=0.002) and vertical septa (P=0.012) in the ED group at 10 and 21 days. Analysis of VEGF showed significant differences among the surgical treatments involving the resting zone, and the proliferative zone for days 1, 6, and 21 (P≤0.02) with greater mean scores present in the fracture (control) group, followed by the ED group; the lowest scores were present in the ES group. PTHrP-R immunolocalization showed significant differences among treatments in the hypertrophic zone at days 6 and 21 (P=0.022 and 0.044, respectively). CONCLUSIONS: On the basis of the type of surgical treatment, results show significant differences in the presence of VEGF (reflecting the vascular bed) in the resting and proliferating zones at days 1, 6, and 21. VEGF localization was less abundant in the ED group (which had more physeal bars), suggesting that lack of vascular ingrowth plays a role in physeal bar formation. CLINICAL RELEVANCE: Basic science data presented here provide insight into the importance of the various regions of the physis and its repair and continued growth after physeal fracture. We suggest that a better understanding of the cellular basis of physeal arrest following physeal fracture may have future relevance for the development of treatments to prevent or correct arrest.

Changes in mRNA gene expression during growth in the femoral head of the young rat.

The rate of physeal growth slows as an animal matures with changes in mRNA gene expression due to the altered cellular activity. To measure the change in gene expression during the juvenile growth period, the femoral head, enclosing the proximal femoral physis, primary spongiosa, and articular cartilage, was collected from both femora of 16 female Sprague-Dawley rats between 4 and 10 weeks of age. One femur of each rat had had a mid-diaphyseal femoral fracture at 4 weeks of age. RNA was extracted and hybridized to 16 Affymetrix Rat Genomic 230 2.0 GeneChip microarrays with probe sets for 31,000 genes of which 18,200 were expressed. Of these, 8002 genes had a significant change in gene expression during growth, about half increasing and half decreasing. These changes included up-regulation with time of genes related to cartilage, blood vessels, osteoprotegerin, osteomodulin, and most ribosomal proteins. There was down-regulation with maturity of genes related to bone, growth-promoting cytokines, G proteins, GTPase-mediated signal transduction factors, cytokine receptors, mitosis, integrin-linked kinase, and the cytoskeleton. In summary, the slowing of growth with maturity was associated with changes in mRNA gene expression in the femoral head for a large number of genes. These changes in gene expression between young and mature rats suggest factors which are important for the support of the rapid linear growth during early life.

Mesenchymal stem cell allograft as a fusion adjunct in one- and two-level anterior cervical discectomy and fusion: a matched cohort analysis.

BACKGROUND CONTEXT: Live mesenchymal stem cell (MSC) allograft-containing allogeneic bone grafts have recently gained popularity and currently account for greater than 17% of all bone grafts and bone graft substitutes used in spinal surgery. Although the claim of cellular bone matrices containing osteogenic cells with osteoinductive properties is attractive, little is known about their clinical success when used in anterior cervical discectomy and fusion (ACDF). PURPOSE: The objective of this study was to report on the radiographic fusion rates in one- and two-level instrumented ACDF using an MSC. STUDY DESIGN/SETTING: This was a retrospective review of prospectively matched cohort of patients with radiologic assessment of fusion as the primary end point. PATIENT SAMPLE: Two matched cohorts of adult patients who underwent ACDF with MSC or standard allograft were included. OUTCOMES MEASURES: The outcome measures included radiographic and clinical evidence of healing at 1 year. METHODS: A consecutive series of 57 patients who underwent a one- or two-level instrumented ACDF procedure between 2010 and 2012 were retrospectively analyzed. All fusion constructs comprised an interbody allograft, an anterior plate, and Osteocel (NuVasive, San Diego, CA, USA). These patients were matched to a control group of 57 patients. RESULTS: Of the 57 cases in both cohorts, 29 (50.9%) were single-level, and 28 (49.1%) were two-level instrumented ACDFs. There were no significant differences in patient age (p=.71), gender, comorbidity burden (Charlson Comorbidity Index [CCI]: 1.95; 2.42, p=.71) or body mass index (p=.79). At the 1-year follow-up, 50 of 57 (87.7%) patients in the Osteocel cohort demonstrated a solid fusion compared with 54 of 57 (94.7%) in the control group (p=.19). Seven (12.3%) patients in the Osteocel cohort were reported as having a failed fusion at 1 year. CONCLUSIONS: This is the first non-industry sponsored study to analyze a matched cohort assessing the 1-year arthrodesis rates associated with a nonstructural MSC allograft in one- and two-level ACDF procedures. Although not statistically significant, patients treated with MSC allografts demonstrated lower fusion rates compared with a matched non-MSC cohort.

Transient soft-tissue edema associated with implantation of increasing doses of rhBMP-2 on an absorbable collagen sponge in an ectopic rat model.

BACKGROUND: Recombinant human bone morphogenetic protein-2 (rhBMP-2) is an osteoinductive protein. However, soft-tissue edema adjacent to the site of rhBMP-2 implantation has been reported. This animal study was designed to examine soft-tissue edema associated with increasing rhBMP-2 doses with implantation on an absorbable collagen sponge (ACS) and with injection directly into muscle. METHODS: Thirty-six Lewis rats received intramuscular implantation of rhBMP-2 on an ACS (Part I) or intramuscular injection of rhBMP-2 solution (Part II). Part-I sites received rhBMP-2/ACS at doses of 0 µg, 30 µg (normal), 129 µg (mid), or 450 µg (high). Part-II sites received rhBMP-2/ACS or rhBMP-2 intramuscular injection at doses of 10 µg (normal) or 150 µg (mid). A previous rat model showed 10 µg to be 100% effective at inducing osseous spinal fusion. In our study, T2-weighted magnetic resonance imaging (MRI) was performed at two and seven days to assess edema volume, and statistical comparisons were carried out with analysis of variance (ANOVA). Cellular response, vascularity, and ossification were examined histologically. RESULTS: Quantitative MRI demonstrated similar peri-implant edema volumes in the control (buffer on an ACS) and normal-dose rhBMP-2 groups. Higher doses resulted in increased edema volume. Edema decreased significantly from two to seven days. Similar capillary densities were observed in all rhBMP-2 groups at two days, and there was dose-dependent increased ossification at seven days. Compared with the rhBMP-2 injection, implantation of the rhBMP-2/ACS resulted in increased edema. This edematous response was transient in all groups. Minimal or no ossification occurred after the rhBMP-2 injections. CONCLUSIONS: Transient peri-implant soft-tissue edema occurred in a dose-dependent manner following implantation of rhBMP-2/ACS in this rat model. The normal dose of rhBMP-2/ACS produced edema similar to that in the controls. Finally, rhBMP-2 solutions injected directly into muscle resulted in minimal soft-tissue edema.

Radiation therapy for heterotopic ossification prophylaxis acutely after elbow trauma: a prospective randomized study.

BACKGROUND: Heterotopic ossification around the elbow can result in pain, loss of motion, and impaired function. We hypothesized that a single dose of radiation therapy could be administered safely and acutely after elbow trauma, could decrease the number of elbows that would require surgical excision of heterotopic ossification, and might improve clinical results. METHODS: A prospective randomized study was conducted at three medical centers. Patients with an intra-articular distal humeral fracture or a fracture-dislocation of the elbow with proximal radial and/or ulnar fractures were enrolled. Patients were randomized to receive either single-fraction radiation therapy of 700 cGy immediately postoperatively (within seventy-two hours) or nothing (the control group). Clinical and radiographic assessment was performed at six weeks, three months, and six months postoperatively. All adverse events and complications were documented prospectively. RESULTS: This study was terminated prior to completion because of an unacceptably high number of adverse events reported in the treatment group. Data were available on forty-five of the forty-eight patients enrolled in this study. When the rate of complications was investigated, a significant difference was detected in the frequency of nonunion between the groups. Of the nine patients who had a nonunion, eight were in the treatment group. The nonunion rate was 38% (eight) of twenty-one patients in the treatment group, which was significantly different from the rate of 4% (one) of twenty-four patients in the control group (p = 0.007). There were no significant differences between the groups with regard to the prevalence of heterotopic ossification, postoperative range of motion, or Mayo Elbow Performance Score noted at the time of study termination. CONCLUSIONS: This study demonstrated that postoperative single-fraction radiation therapy, when used acutely after elbow trauma for prophylaxis against heterotopic ossification, may play a role in increasing the rate of nonunion at the site of the fracture or an olecranon osteotomy. The clinical efficacy of radiation therapy could not be determined on the basis of the sample size. Further research is needed to determine the role of limited-field radiation for prophylaxis against heterotopic ossification after elbow trauma.

Evidence for overgrowth after midfemoral fracture via increased RNA for mitosis.

Middiaphyseal femoral fractures in children and young rats stimulate linear femoral growth, a phenomenon commonly attributed to increased vascularity. To test for changes in mRNA expression of genes related to blood vessels, nerve fibers, cartilage, bone, and cell metabolism, we measured mRNA gene expression for all known rat genes in the physis at various times after diaphyseal fracture. Female Sprague-Dawley rats, 4 weeks of age at surgery, were subjected to a unilateral, simple, transverse, middiaphyseal femoral fracture stabilized with an intramedullary rod. At 0 (intact), 0.1, 0.4, 1, 2, 3, 4, and 6 weeks after fracture, the femoral head with the proximal physis was collected from fractured and intact femora. The RNA was extracted, processed to biotinlabeled cRNA, and hybridized to Affymetrix Rat 230 2.0 GeneChip microarrays. Transcripts from fracture-induced lengthening of the injured femora were compared to those of the intact contralateral femur. In the proximal physis, transcripts related to blood vessels and cartilage formation were down-regulated by fracture. Transcripts related to bone remodeling, nerve axon elongation, cell division, and protein synthesis were up-regulated by fracture. The data support increased mitotic activity in the physis after a midshaft fracture and not increased vascularity.

Vertebral endplate architecture and vascularization: application of micro-computerized tomography, a vascular tracer, and immunocytochemistry in analyses of disc degeneration in the aging sand rat.

STUDY DESIGN: Lower lumbar vertebral endplates from young and old sand rats were assessed in an Institutional Animal Care and Use Committee approved study for architectural endplate features using micro-computerized tomography (CT) 3-dimensional (3D) models and vascularization studies by an in vivo vascular tracer or immunocytochemical identification of blood vessels. OBJECTIVE: To assess endplate porosity and vascularization using microCT architectural analysis, an in vivo vascular tracer, and immunocytochemical identification of blood vessels in the endplate. SUMMARY OF THE BACKGROUND DATA: The vertebral endplates, also called cartilage endplates, form the superior and inferior, or cranial and caudal, boundaries of the disc. In the human being and sand rat, the cartilaginous endplate undergoes calcification with aging and is replaced by bone. Endplate sclerosis has long been thought to play a role in disc degeneration by decreasing nutrient availability to the disc, but this is still poorly understood. Previous work has identified increasing bone mineral density with aging and disc degeneration in the sand rat model. METHODS: microCT models of the lower lumbar endplates of vertebrae at L5-6 and L6-7 were constructed from 6 younger (mean age 11 months) and 21 older (mean age 25.6 months) sand rats. Architectural features were scored on a semiquantitative scale for smoothness of the endplate face, irregularities on the endplate margin, and endplate thickness. There were 2 smaller sets of animals (n = 18) evaluated for endplate vascularity following in vivo injection of a fluorescent vascular tracer or by the use of immunocytochemistry to identify blood vessels. RESULTS: microCT revealed a solid bony surface to the endplate, which was not penetrated by vasculature; with aging/disc degeneration, there was roughening and pitting of the plate surface, and the development of irregular margins. In L5-6 and L6-7, sites of prominent disc degeneration evident on radiographs, the proportion of abnormalities in surface smoothness, margin irregularity, and endplate thickening were all statistically significant in both younger and older animals (P < or = 0.0027). More severe changes were evident in the caudal versus cranial endplate surfaces. Histologic study of vascular tracer showed that there was no penetration of the disc by vascular supply from the endplate; this was verified by immunocytochemical identification of blood vessels. The canal system within the endplate was a complex 3D interconnected network. CONCLUSIONS: Findings show that disc degeneration in the sand rat occurs concomitantly with marked architectural bony changes on the endplate face, including loss of smoothness and development of irregular bony margins. Vascular connections were not present between the endplate and disc; this was verified with microCT studies, in vivo vascular tracers, and traditional immunocytochemistry. The canal system within the imaged endplate was revealed to consist of a complex 3D interconnected network.