Quantitative imaging of murine osteoarthritic cartilage by phase-contrast micro-computed tomography.

OBJECTIVE: The mouse is an optimal model organism in which gene-environment interactions can be used to study the pathogenesis of osteoarthritis (OA). The gold standard for arthritis research in mice is based on histopathology and immunohistochemistry, which are labor-intensive, prone to sampling bias and technical variability, and limited in throughput. The aim of this study was to develop a new technique that assesses mouse cartilage by integrating quantitative volumetric imaging techniques. METHODS: A novel mouse model of OA was generated by cruciate ligament transection (CLT) and evaluated by histopathology and immunohistochemistry. Knee joint specimens were then imaged using a new technique that combines high-resolution micro-computed tomography (micro-CT) and phase-contrast optics followed by quantitative analyses. A comparative analysis was also performed in a previously established mouse model of OA generated by destabilization of the medial meniscus (DMM). RESULTS: Phase-contrast micro-CT achieved cellular resolution of chondrocytes and quantitative assessment of parameters such as articular cartilage volume and surface area. In mouse models of OA generated by either CLT or DMM, we showed that phase-contrast micro-CT distinguished control and OA cartilage by providing quantitative measures with high reproducibility and minimal variability. Features of OA at the cellular or tissue level could also be observed in images generated by phase-contrast micro-CT. CONCLUSION: We established an imaging technology that comprehensively assessed and quantified the 2-dimensional and 3-dimensional changes of articular cartilage. Application of this technology will facilitate the rapid and high-throughput assessment of genetic and therapeutic models of OA in mice.

Prospective randomized phase II Trial of accelerated reepithelialization of superficial second-degree burn wounds using extracorporeal shock wave therapy.

BACKGROUND: As extracorporeal shock wave therapy (ESWT) can enhance healing of skin graft donor sites, this study focused on shock wave effects in burn wounds. METHODS: A predefined cohort of 50 patients (6 with incomplete data or lost to follow-up) with acute second-degree burns from a larger study of 100 patients were randomly assigned between December 2006 and December 2007 to receive standard therapy (burn wound debridement/topical antiseptic therapy) with (n = 22) or without (n = 22) defocused ESWT (100 impulses/cm at 0.1 mJ/mm) applied once to the study burn, after debridement. Randomization sequence was computer-generated, and patients were blinded to treatment allocation. The primary endpoint, time to complete burn wound epithelialization, was determined by independent, blinded-observer. A worst case scenario was applied to the missing cases to rule out the impact of withdrawal bias. RESULTS: Patient characteristics across the 2 study groups were balanced (P > 0.05) except for older age (53 ± 17 vs. 38 ± 13 years, P = 0.002) in the ESWT group. Mean time to complete (≥95%) epithelialization (CE) for patients that did and did not undergo ESWT was 9.6 ± 1.7 and 12.5 ± 2.2 days, respectively (P < 0.0005). When age (continuous variable) and treatment group (binary) were examined in a linear regression model to control the baseline age imbalance, time to CE, age was not significant (P = 0.33) and treatment group retained significance (P < 0.0005). Statistical significance (P = 0.001) was retained when ESWT cases with missing follow-up were assigned the longest time to CE and when controls with missing follow-up were assigned the shortest time to CE. CONCLUSIONS: In this randomized phase II study, application of a single defocused shock wave treatment to the superficial second-degree burn wound after debridement/topical antiseptic therapy significantly accelerated epithelialization. This finding warrants confirmation in a larger phase III trial (ClinicalTrials.gov identifier: NCT01242423).

Cell-based gene therapy for repair of critical size defects in the rat fibula.

More than a decade has passed since the first experiments using adenovirus-transduced cells expressing bone morphogenetic protein 2 were performed for the synthesis of bone. Since this time, the field of bone gene therapy has tackled many issues surrounding safety and efficacy of this type of strategy. We present studies examining the parameters of the timing of bone healing, and remodeling when heterotopic ossification (HO) is used for bone fracture repair using an adenovirus gene therapy approach. We use a rat fibula defect, which surprisingly does not heal even when a simple fracture is introduced. In this model, the bone quickly resorbs most likely due to the non-weight bearing nature of this bone in rodents. Using our gene therapy system robust HO can be introduced at the targeted location of the defect resulting in bone repair. The HO and resultant bone healing appeared to be dose dependent, based on the number of AdBMP2-transduced cells delivered. Interestingly, the HO undergoes substantial remodeling, and assumes the size and shape of the missing segment of bone. However, in some instances we observed some additional bone associated with the repair, signifying that perhaps the forces on the newly forming bone are inadequate to dictate shape. In all cases, the HO appeared to fuse into the adjacent long bone. The data collectively indicates that the use of BMP2 gene therapy strategies may vary depending on the location and nature of the defect. Therefore, additional parameters should be considered when implementing such strategies.

Human Peripheral Nerve-Derived Pluripotent Cells Can Be Stimulated by In Vitro Bone Morphogenetic Protein-2.

We have recently identified a population of cells within the peripheral nerves of adult rodent animals (mice and rats) that can respond to Bone Morphogenetic Protein-2 (BMP-2) exposure or physical injury to rapidly proliferate. More importantly, these cells exhibited embryonic differentiation potentials that could be induced into osteoblastic and endothelial cells in vitro. The current study examined human nerve specimens to compare and characterize the cells after BMP-2 stimulation. Fresh pieces of human nerve tissue were minced and treated with either BMP-2 (750 ng/mL) or a PBS vehicle for 12 h at 37 °C, before being digested in 0.2% collagenase and 0.05% trypsin-EDTA. Isolated cells were cultured in a restrictive stem cell medium. Significantly more cells were obtained from the nerve pieces with the BMP-2 treatment in comparison with the PBS vehicle controls. Cell colonies started to form at Day 3. Expressions of the four transcription factors, namely, Klf4, c-Myc, Sox2, and Oct4, were confirmed at both the transcriptional and translational levels. The cells can be maintained in the stem cell culture medium for at least 6 weeks without changing their morphology. When the cells were transferred to a fibroblast growth medium, dispersed spindle-shaped motile cells were noted and became fibroblast activated protein-α (FAP) positive with immunocytochemistry staining. The data suggest that human peripheral nerve tissue also contains a population of cells that can respond to BMP-2 and express Klf4, Sox2, cMyc, and Oct4-the four transcription factors driving cell pluripotency. These cells are able to differentiate into FAP-positive fibroblasts. In summary, in human peripheral nerves also reside a population of quiescent cells with pluripotency potential that may be the same cells as rodent nerve-derived adult stem (NEDAPS) cells. It is proposed that these cells are possibly at the core of a previously unknown natural mechanism for healing an injury.

Cervical fusion for treatment of degenerative conditions: development of appropriate use criteria.

BACKGROUND CONTEXT: High quality evidence is difficult to generate, leaving substantial knowledge gaps in the treatment of spinal conditions. Appropriate use criteria (AUC) are a means of determining appropriate recommendations when high quality evidence is lacking. PURPOSE: Define appropriate use criteria (AUC) of cervical fusion for treatment of degenerative conditions of the cervical spine. STUDY DESIGN/SETTING: Appropriate use criteria for cervical fusion were developed using the RAND/UCLA appropriateness methodology. Following development of clinical guidelines and scenario writing, a one-day workshop was held with a multidisciplinary group of 14 raters, all considered thought leaders in their respective fields, to determine final ratings for cervical fusion appropriateness for various clinical situations. OUTCOME MEASURES: Final rating for cervical fusion recommendation as either "Appropriate," "Uncertain" or "Rarely Appropriate" based on the median final rating among the raters. METHODS: Inclusion criteria for scenarios included patients aged 18 to 80 with degenerative conditions of the cervical spine. Key modifiers were defined and combined to develop a matrix of clinical scenarios. The median score among the raters was used to determine the final rating for each scenario. The final rating was compared between modifier levels. Spearman's rank correlation between each modifier and the final rating was determined. A multivariable ordinal regression model was fit to determine the adjusted odds of an "Appropriate" final rating while adjusting for radiographic diagnosis, number of levels and symptom type. Three decision trees were developed using decision tree classification models and variable importance for each tree was computed. RESULTS: Of the 263 scenarios, 47 (17.9 %) were rated as rarely appropriate, 66 (25%) as uncertain and 150 (57%) were rated as appropriate. Symptom type was the modifier most strongly correlated with the final rating (adjusted ρ2 = 0.58, p<.01). A multivariable ordinal regression adjusting for symptom type, diagnosis, and number of levels and showed high discriminative ability (C statistic = 0.90) and the adjusted odds ratio (aOR) of receiving a final rating of "Appropriate" was highest for myelopathy (aOR, 7.1) and radiculopathy (aOR, 4.8). Three decision tree models showed that symptom type and radiographic diagnosis had the highest variable importance. CONCLUSIONS: Appropriate use criteria for cervical fusion in the setting of cervical degenerative disorders were developed. Symptom type was most strongly correlated with final rating. Myelopathy or radiculopathy were most strongly associated with an "Appropriate" rating, while axial pain without stenosis was most associated with "Rarely Appropriate."

Heterotopic ossification has some nerve.

Heterotopic ossification, defined as the formation of bone in abnormal anatomic locations, can be clinically insignificant or devastating and debilitating, depending on the site and duration of new bone formation. There are many causes of heterotopic ossification (HO), including soft tissue trauma, central nervous system injury, vasculopathies, arthropathies, and inheritance. One of the least understood components of HO is the interaction of the peripheral nervous system with the induction of this process. Recent work has shown that, upon traumatic injury, a cascade of events termed neurogenic inflammation is initiated, which involves the release of neuropeptides, such as substance P and calcitonin gene related peptide. Release of these peptides ultimately leads to the recruitment of activated platelets, mast cells, and neutrophils to the injury site. These cells appear to be involved with both remodeling of the nerve, as well as potentially recruiting additional cells from the bone marrow to the injury site. Further, sensory neurons stimulated at the injury site relay local information to the brain, which can then redirect neuroendocrine signaling in the hypothalamus towards repair of the injured site. While numerous studies have highlighted the important role of nerve-derived signals, both central and peripheral, in the regulation of normal bone remodeling of the skeleton,1 this review focuses on the role of the local, peripheral nerves in the formation of heterotopic bone. We concentrate on the manner in which local changes in bone morphogenetic protein (BMP) expression contribute to a cascade of events within the peripheral nerves, both sensory and sympathetic, in the immediate area of HO formation.

Effect of BMP-2 Adherent to Resorbable Sutures on Cartilage Repair: A Rat Model of Xyphoid Process.

Meniscal tears are often seen in orthopedic practice. The current strategy for meniscal repair has only had limited success with a relatively high incidence of re-operative rate. This study evaluates the therapeutic effects of Bone morphogenetic protein-2 (BMP-2) soaked sutures for cartilage repair, using a rat model of xyphoid healing. Vicryl-resorbable sutures were presoaked in BMP-2 solutions prior to animal experimentation. Rat xyphoid process (an avascular hyaline cartilage structure) was surgically ruptured followed by repair procedures with regular suture or with sutures that were pre-soaked in BMP-2 solutions. In vitro assessment indicated that presoaking the Vicryl-resorbable sutures with 10 µg/mL BMP-2 resulted in a sustained amount of the growth factor release up to 7 days. Histological analysis suggested that application of this BMP-2 soaked suture on the rat xyphoid process model significantly improved the avascular cartilage healing compared to non-soaked control sutures. In conclusion, data here confirm that the rat xyphoid process repair is a reproducible and inexpensive animal model for meniscus and other cartilage repair. More importantly, coating of BMP-2 on sutures appears a potential avenue to improve cartilage repair and regeneration. Further study is warranted to explore the molecular mechanisms of this strategy.

Prevention and management of osteoporosis and osteoporotic fractures in persons with a spinal cord injury or disorder: A systematic scoping review.

Objectives: The primary objective was to review the literature regarding methodologies to assess fracture risk, to prevent and treat osteoporosis and to manage osteoporotic fractures in SCI/D.Study Design: Scoping review.Settings/Participants: Human adult subjects with a SCI/D.Outcome measures: Strategies to identify persons with SCI/D at risk for osteoporotic fractures, nonpharmacological and pharmacological therapies for osteoporosis and management of appendicular fractures.Results: 226 articles were included in the scoping review. Risk of osteoporotic fractures in SCI is predicted by a combination of DXA-defined low BMD plus clinical and demographic characteristics. Screening for secondary causes of osteoporosis, in particular hyperparathyroidism, hyperthyroidism, vitamin D insufficiency and hypogonadism, should be considered. Current antiresorptive therapies for treatment of osteoporosis have limited efficacy. Use of surgery to treat fractures has increased and outcomes are good and comparable to conservative treatment in most cases. A common adverse event following fracture was delayed healing.Conclusions: Most of the research in this area is limited by small sample sizes, weak study designs, and significant variation in populations studied. Future research needs to address cohort definition and study design issues.

Degenerative lumbar spinal stenosis: an evidence-based clinical guideline for the diagnosis and treatment of degenerative lumbar spinal stenosis.

BACKGROUND CONTEXT: The objective of the North American Spine Society (NASS) evidence-based clinical guideline for the diagnosis and treatment of degenerative lumbar spinal stenosis (DLSS) is to provide evidence-based recommendations to address key clinical questions surrounding the diagnosis and treatment of DLSS. The guideline is intended to reflect contemporary treatment concepts for symptomatic DLSS as reflected in the highest quality clinical literature available on this subject as of April 2006. The goals of the guideline recommendations are to assist in delivering optimum, efficacious treatment, and functional recovery from this spinal disorder. PURPOSE: To provide an evidence-based tool that assists practitioners in improving the quality and efficiency of care delivered to patients with DLSS. STUDY DESIGN/SETTING: Evidence-based clinical guideline. METHODS: This report is from the Spinal Stenosis Work Group of the NASS Clinical Guidelines Committee. The work group comprised medical, diagnostic, interventional, and surgical spinal care specialists, all of whom were trained in the principles of evidence-based analysis. In the development of this guideline, the work group arrived at a consensus definition of a working diagnosis of lumbar spinal stenosis by use of a modification of the nominal group technique. Each member of the group formatted a series of clinical questions to be addressed by the group and the final list of questions agreed on by the group is the subject of this report. A literature search addressing each question and using a specific literature search protocol was performed on English language references found in MEDLINE, EMBASE (Drugs and Pharmacology), and four additional, evidence-based, databases. The relevant literature to answer each clinical question was then independently rated by at least two reviewers using the NASS-adopted standardized levels of evidence. An evidentiary table was created for each of the questions. Any discrepancies in evidence levels among the initial raters were resolved by at least two additional members' review of the reference and independent rating. Final grades of recommendation for the answer to each clinical question were arrived at in face-to-face meetings among members of the work group using the NASS-adopted standardized grades of recommendation. When Levels I to IV evidence was insufficient to support a recommendation to answer a specific clinical question, expert consensus was arrived at by the work group through the modified nominal group technique and is clearly identified as such in the guideline. RESULTS: Eighteen clinical questions were asked, addressing issues of prognosis, diagnosis, and treatment of DLSS. The answers to these 18 clinical questions are summarized in this document along with their respective levels of evidence and grades of recommendation in support of these answers. CONCLUSIONS: A clinical guideline for DLSS has been created using the techniques of evidence-based medicine and using the best available evidence as a tool to aid both practitioners and patients involved with the care of this disease. The entire guideline document including the evidentiary tables, suggestions for future research, and all references is available electronically at the NASS Web site (www.spine.org) and will remain updated on a timely schedule.

Morphology and Clinical Relevance of Vertebral Endplate Changes Following Limited Lumbar Discectomy With or Without Bone-anchored Annular Closure.

STUDY DESIGN: Post hoc analysis of a randomized controlled trial. OBJECTIVE: To characterize the morphology and clinical relevance of vertebral endplate changes (VEPC) following limited lumbar discectomy with or without implantation of a bone-anchored annular closure device (ACD). SUMMARY OF BACKGROUND DATA: Implantation of an ACD following limited lumbar discectomy has shown promise in reducing the risk of recurrent herniation in patients with large annular defects. However, the interaction between the ACD and the lumbar endplate over time is not well understood. METHODS: Patients undergoing limited lumbar discectomy with large postsurgical annular defects were randomized intraoperatively to receive additional ACD implantation or limited lumbar discectomy only (Controls). VEPC morphology, area, and volume were assessed with low-dose computed tomography preoperatively and at 1 and 2 years follow-up. RESULTS: Of 554 randomized patients, the as-treated population consisted of 550 patients (267 ACD, 283 Controls). VEPC were preoperatively identified in 18% of patients in the ACD group and in 15% of Controls. At 2 years, VEPC frequency increased to 85% with ACD and 33% in Controls. Device- or procedure-related serious adverse event (8% vs. 17%, P = 0.001) and secondary surgical intervention (5% vs. 13%, P < 0.001) favored the ACD group over Controls. In the ACD group, clinical outcomes were comparable in patients with and without VEPC at 2 years follow-up. In the Control group, patients with VEPC at 2 years had higher risk of symptomatic reherniation versus patients without VEPC (35% vs. 19%, P < 0.01) CONCLUSION.: In patients with large annular defects following limited lumbar discectomy, additional implantation with a bone-anchored ACD reduces risk of postoperative complications despite a greater frequency of VEPC. VEPC were associated with higher risk of symptomatic reherniation in patients treated with limited lumbar discectomy, but not in those who received additional ACD implantation. LEVEL OF EVIDENCE: 2.

Efficient and rapid osteoinduction in an immune-competent host.

Osteoinductive systems to induce targeted rapid bone formation hold clinical promise, but development of technologies for clinical use that must be tested in animal models is often a difficult challenge. We previously demonstrated that implantation of human cells transduced with Ad5F35BMP2 to express high levels of bone morphogenetic protein-2 (BMP2) resulted in rapid bone formation at targeted sites. Inclusion of human cells in this model precluded us from testing this system in an immune-competent animal model, thus limiting information about the efficacy of this approach. Here, for the first time we demonstrate the similarity between BMP2-induced endochondral bone formation in a system using human cells in an immune-incompetent mouse and a murine cell-based BMP2 gene therapy system in immune-competent animals. In both cases the delivery cells are rapidly cleared, within 5 days, and in neither case do they appear to contribute to any of the structures forming in the tissues. Endochondral bone formation progressed through a highly ordered series of stages that were both morphologically and temporally indistinguishable between the two models. Even longterm analysis of the heterotopic bone demonstrated similar bone volumes and the eventual remodeling to form similar structures. The results suggest that the ability of BMP2 to rapidly induce bone formation overrides contributions from either immune status or the nature of delivery cells.

Oxygen tension directs chondrogenic differentiation of myelo-monocytic progenitors during endochondral bone formation.

Synthesis of bone requires both essential progenitors to form the various structures and the correct microenvironment for their differentiation. To identify these factors, we have used a system that exploits bone morphogenetic protein's ability to induce endochondral bone formation rapidly. One of the earliest events observed was the influx and proliferation of fibroblastic cells that express both vascular smooth muscle cell markers, alpha smooth muscle actin (alpha SMA), smooth muscle myosin heavy chain, and the monocytic marker CD68. The expression of these factors was lost by days 4 to 5, coincident with the up-regulation of Sox9 and the appearance of chondrocytes. Studies with a cyclization recombination (Cre)/lox system, in which a myeloid-specific promoter driving Cre recombinase can irreversibly unblock expression of beta-galactosidase only in cells of myeloid origin, showed specific activity in the newly formed chondrocytes. These results suggest that early chondrocyte progenitors are of myeloid origin. Simultaneous with this recruitment, we determined that a numbers of these cells were in a hypoxic state, indicative of a low-oxygen environment. The cells in the hypoxic regions were undergoing chondrogenesis, whereas cells in adjacent normoxic regions appeared to be assembling into new vessels, suggesting that the oxygen microenvironment is critical for establishment of the cartilage.

Intraosseous innervation of the human patella: a histologic study.

BACKGROUND: The soft tissue structures surrounding the human knee joint have been the subject of extensive anatomic study. The detailed histologic findings within the bone of the human patella, however, have not been systematically studied. While the nerves supplied to the periarticular soft tissues have been very well documented, the nerves supplied to the interior of the bony patella have never been described. HYPOTHESIS: This study tests the hypothesis that the patella contains an intraosseous nerve network. Further, the authors investigate the anatomic location of these intraosseous nerves to better understand their possible clinical relevance. STUDY DESIGN: Descriptive laboratory study. METHODS: Ten matched pairs of cadaveric patellae (left and right patellae from the same individual; 20 total) were prepared for evaluation by hematoxylin and eosin staining using a technique that allows the creation of complete, large histologic sections of individual patellae. The matched specimens were dissected free of soft tissue and then sectioned using a diamond-wafering saw into 3-mm sagittal (left patella) and transverse (right patella) sections. Sections were then decalcified and whole-mounted into paraffin blocks for further sectioning using a large-format microtome. All 20 specimens were prepared for evaluation. Age at death averaged 80 years (range, 64-91). All specimens demonstrated at least grade II chondromalacia. RESULTS: Nineteen of 20 (95%) specimens demonstrated intraosseous nerves. Of 248 sections studied, 116 (47%) demonstrated intraosseous nerves, with 227 individual nerves identified. The density of intraosseous nerves was greatest in the medial and central portions of the patella, with a significant paucity identified laterally. CONCLUSION: The primary intraosseous innervation of the patella derives from a medially based neurovascular bundle. CLINICAL RELEVANCE: A better understanding of the nerves within the human bony patella may improve understanding the patho-physiology of anterior knee pain syndromes.

Initial intervertebral stability after anterior cervical discectomy and fusion with plating.

BACKGROUND CONTEXT: Anterior cervical locking plates are currently used to provide secure fixation, which appears to help promote fusion, although the details of the in vivo biomechanical effects of plating on the spine are unknown. PURPOSE: To determine if any motion was present initially in the plated anterior cervical discectomy and fusion (ACDF) and, if so, where the motion occurred. STUDY DESIGN: A cohort of patients that were part of a prospective study on postoperative changes after cervical fusion were evaluated for motion at the fusion site about 2 weeks after ACDF. METHODS: Forty-eight segments in 27 patients undergoing ACDF with a cervical plate were evaluated. The patients underwent flexion and extension radiography approximately 2 weeks after surgery. Intervertebral motion at the fusion site was evaluated with validated quantitative motion analysis. RESULTS: Motion was perceived at 29 levels in 18 patients. The sources of motion were either actual bending of the plate itself, motion at the screw-bone interface, or screw-plate interface. CONCLUSIONS: In many patients, anterior cervical plating after ACDF does not eliminate motion at the fusion site. Depending on the quality of bone, type of bone graft, and nature of the injury, this information may influence decisions regarding implants and postoperative bracing.

Ablation of the basivertebral nerve for treatment of back pain: a clinical study.

BACKGROUND CONTENT: Lumbar axial back pain arising from degenerative disc disease continues to be a challenging clinical problem whether treated with nonsurgical management, local injection, or motion segment stabilization and fusion. PURPOSE: The purpose of this study was to determine the efficacy of intraosseous basivertebral nerve (BVN) ablation for the treatment of chronic lumbar back pain in a clinical setting. STUDY DESIGN: Patients meeting predefined inclusion or exclusion criteria were enrolled in a study using radiofrequency energy to ablate the BVN within the vertebral bodies adjacent to the diagnosed level. Patients were evaluated at 6 weeks, and 3, 6, and 12 months postoperatively. PATIENT SAMPLE: Seventeen patients with chronic, greater than 6 months, low back pain unresponsive to at least 3 months of conservative care were enrolled. Sixteen patients were treated successfully following screening using magnetic resonance imaging finding of Modic type I or II changes and positive confirmatory discography to determine the affected levels. The treated population consisted of eight male and eight female patients; the mean age was 48 years (34-66 years). OUTCOME MEASURES: Self-reported outcome measures were collected prospectively at each follow-up interval. Measures included the Oswestry Disability Index (ODI), visual analogue scale score, and Medical Outcomes Trust 36-Item Short-Form Health Survey (SF-36). MATERIALS AND METHODS: This is an industry-sponsored study to evaluate the effectiveness of intraosseous nerves in the treatment of chronic back pain. Consented and enrolled patients underwent ablation of the BVN using radiofrequency energy (INTRACEPT System, Relievant Medsystems, Inc, Redwood City, CA, USA) guided in a transpedicular or extrapedicular approach. Preoperative planning determined targeted ablation zone and safety zones. RESULTS: Mean baseline ODI of the treated cohort was 52±13, decreasing to a mean of 23±21 at 3 months follow-up (p<.001). The statistically significant improvement in ODI observed at 3 months was maintained through the 12-month follow-up. The mean baseline visual analogue scale score decreased from 61±22 to 45±35 at 3 months follow-up (p<.05), and the mean baseline physical component summary increased from 34.5±6.5 to 41.7±12.4 at 3 months follow-up (p=.03). CONCLUSION: Ablation of the BVN for the treatment of chronic lumbar back pain significantly improves patients' self-reported outcome early in the follow-up period; the improvement persisted throughout the 1-year study period.

Quiescent pluripotent stem cells reside within murine peripheral nerves that can be stimulated to proliferate by recombinant human bone morphogenic protein 2 or by nerve trauma.

BACKGROUND: The clinical use of recombinant human bone morphogenic protein 2 (rhBMP-2, Infuse) has been associated with nerve-related complications including new-onset sciatica, and retrograde ejaculation. PURPOSE: To better understand the interaction of rhBMP-2 and peripheral nerves with the intent of making procedures safer. STUDY DESIGN/SETTING: Using a mouse model to examine the direct effect of diluted rhBMP-2 (Infuse) on murine sciatic nerves. METHODS: Animal studies were approved by the Institutional Animal Care and Use Committee. Balb/c mouse sciatic nerves were surgically exposed and 60 ng (in 10 µL) of rhBMP-2 was applied to the nerve. In separate experiments, the sciatic nerves were subjected to mechanical compression using forceps (and not exposed to rhBMP-2). The third group of mice received direct injection of the same amount of rhBMP-2, or sterile saline as a control, into the hamstring area of the posterior thigh without surgery. Mouse limbs with intact sciatic nerve were collected at 24, 48, or 72 hours after treatment for histology processing. A separate set of identically treated sciatic nerves were retrieved from mice at the same time points and cells were isolated by collagenase and trypsin digestion. The isolated cells were cultured in a stem cell medium containing 20% knockout serum and human leukemia inhibitory factor. Immunohistochemical or immunofluorescent cell stains against KLF4, Sox2, c-Myc, and Oct4 were performed on the mouse tissue sections and cell culture slides. In addition, real-time polymerase chain reaction (PCR) was performed to quantify the mRNA expression profiles of the stem cell marker genes in cultured cells. RESULTS: Profound morphological changes of the mouse sciatic nerves were noted after exposure to rhBMP-2, with a rapid and robust cell proliferation within the nerves followed by migration of these cells into surrounding tissue. Immunohistochemical stain revealed strong nuclear stains of KLF4, Sox2, Oct4, and c-Myc on the overwhelming majority of these proliferating cells in the nerve. Intramuscular injections of rhBMP-2 or willful physical compression of the nerves showed similar cell proliferation effects as the direct application of Infuse to the sciatic nerve. The cells in stem cell culture medium grew steadily without feeder cells and appeared fairly uniform. They were adherent to substrate and were motile. Double fluorescent staining on the cells indicated colocalizationof all pairs of the four stem cell markers in the cell nuclei. Real-time PCR confirmed the strong mRNA expressions of KLF4, Sox2, Oct4, and c-Myc in these isolated cells. CONCLUSION: Exposure to BMP-2 causes a marked proliferation of previously quiescent cells within peripheral nerves. These cells simultaneously express KLF4, Sox2, Oct4, and c-Myc, the transcription factors that confer embryonic pluripotency. Work described in the companion paper reveals some of the differentiation capacity of the cells and their likely clinical significance. In addition, the effects of direct exposure of nerves to rhBMP-2 as described here should clearly illuminate the mechanism of BMP-2-related nerve complications. We would suggest that the use of this agent in proximity to known neural structures should only be done with extreme caution.

Differentiation of nerve-derived adult pluripotent stem cells into osteoblastic and endothelial cells.

BACKGROUND CONTEXT: Stem cell-involved tissue engineering has gained dramatic attention as a therapeutic strategy for tissue regeneration including bone repair. However, the currently available possibilities to use embryonic stem cells and induced pluripotent stem cells (iPCs) face potential ethical issues, as well as risks of malignant transformation and immune rejection. Recently identified peripheral nerve-derived adult pluripotent stem cells (NEDAPS) that quickly proliferate after exposure to bone morphogenetic protein-2 (BMP-2) or nerve trauma and exhibit many embryonic stem cell characteristics may provide an attractive source cells for a variety of regenerative therapies. PURPOSE: The study aimed to examine the differentiation potential of the NEDAPS cells into osteoblastic cells and endothelial cells. STUDY DESIGN/SETTING: An in vitro investigation was undertaken to induce mouse NEDAPS cells into the phenotypes of osteoblastic and endothelial cells. METHODS: NEDAPS cells were isolated from low-dose BMP-2-exposed mouse sciatic nerves by collagenase and trypsin extraction. The cells were cultured in a stem cell maintenance medium, and the expression of KLF4, Sox2, c-Myc, and Oct4 before differentiation was confirmed. The cells were then subcultured in a complete osteogenic cell induction medium or endothelial cell growth medium, respectively, at 37°C and 5%CO2 atmosphere. Histologic, morphologic, and molecular assessments were performed 7 days later. RESULTS: The cells propagated in complete osteogenic medium for 7 days showed strong staining for type I collagen and alkaline phosphatase, suggesting the structural and functional properties of the osteoblastic cells. Further, real-time polymerase chain reaction (RT-PCR) revealed a significant expression of the osteoblast markers osteocalcin, osteopontin, and type I collagen. Similarly, the cells in endothelial growth medium were successfully differentiated into cobblestone-shaped endothelial cells expressing vascular endothelial growth factor (VEGF) receptors Flk-1 and Flt-1 demonstrated by RT-PCR. CONCLUSIONS: NEDAPS cells are readily induced to osteoblastic and endothelial cells, suggesting therapeutic potential for bone repair and other regenerative therapies.

Biomechanical evaluation of CIBOR spine interbody fusion device.

BACKGROUND: The CIBOR PEEK spinal interbody fusion device is an anterior lumbar interbody fusion construct with a hollow center designed to accommodate an osteoinductive carbon foam insert to promote bony ingrowth to induce fusion where rigid stabilization is needed. METHODS: Three different sizes of the device were investigated. Part-I: implants were tested under axial compression and rotation using polyurethane foam blocks. Part-II: simulated 2-legged stance using cadaveric specimen using the L5-S1 lumbar spine segment. Part-III: a survey feedback form was used to investigate two orthopedic surgeons concern regarding the implant. RESULTS: In Part-I, the subsidence hysteresis under axial compression loading was found to be statistical significant difference between these three implant sizes. It was noted that the implants had migration as rotation applied, and the amount of subsidence was a factor of the axial compression loads applied. In Part-II, a minor subsidence and carbon foam debris were observed when compared to each implant size. Poor contact surface of the implant with the end plates of the L5 or S1 vertebrae from the anterior view under maximum loads was observed; however, the implant seemed to be stable. Each surgeon has their own subjective opinion about the CIBOR implant. DISCUSSION: Two out of the three different sizes of the device (medium and large sizes) provided appropriate rigid stabilization at the physiological loads. Neither orthopedic surgeon was 100% satisfied with overall performance of the implant, but felt potential improvement could be made. CLINICAL RELEVANCE: This study indicates an option for operative treatment of spine interbody fusion, as the CIBOR spine interbody fusion device has a hollow center. This hollow center is designed to accommodate a carbon foam insert to promote bony ingrowth. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1157-1168, 2017.

Force-mediated dissociation of proteoglycan aggregate in articular cartilage.

Proteoglycan aggregate is the primary component in articular cartilage responsible for resisting compressive loading. It consists of a core molecule of hyaluronan and a number of side chains of aggrecan bound to hyaluronan non-covalently. The loss of aggrecan from articular cartilage is considered to be a major factor in the development of osteoarthritis. Though enzymatic digestion of aggrecan is believed to be responsible for the release of aggrecan from osteoarthritic cartilage, other mechanisms, such as direct force-mediated detachment of aggrecan from hyaluronan may also be involved. In this study, the rupture force of the single bond between hyaluronan and aggrecan in articular cartilage was directly quantified using experimental measurement and Monte Carlo simulation. Low rupture force of this bond, as determined in this study suggested a possible direct force-mediated detachment of aggrecan from proteoglycan aggregate in osteoarthritic cartilage.

Therapeutic effects of naringin on degenerative human nucleus pulposus cells for discogenic low back pain.

BACKGROUND: Over half the population of the world will suffer from moderate or severe low back pain (LBP) during their life span. Studies have shown that naringin, a major flavonoid in grapefruit and an active compound extracted from a Chinese herbal medicine (Rhizoma Drynariae) possesses many pharmacological effects. PURPOSE: The aim of this study was to evaluate the influence of naringin on the growth of degenerative human nucleus pulposus (NP) cells, and its repair effects on protein and gene expressions of the cells. STUDY DESIGN/SETTING: This was an in vitro investigation of the human NP cells isolated from degenerated intervertebral discs that were interacted with various concentrated of naringin. METHOD: This study was exempted by the institutional Human Subjects Committee-2, University of Kansas School of Medicine-Wichita. Degenerative human NP cells were isolated from intervertebral discs of patients with discogenic LBP and cultured at 37°C with 5% CO2. The proliferation of NP cells was determined following treatment with various concentrations of naringin. The protein expressions of tumor necrosis factor-α (TNF-α) and Bone morphogenetic protein 2 (BMP-2) were tested using enzyme-linked immunosorbent assay. Aggrecan and type II collagen levels were measured by immunohistological staining. Further examination of the gene expression of aggrecan, Sox6, and MMP3 was performed after intervention with naringin for 3 days. RESULTS: The human NP cells were successfully propagated in culture and stained positive with toluidine blue staining. Naringin effectively enhanced the cell proliferation at an optimal concentration of 20 µg/mL. Naringin treatment resulted in significant inhibition of TNF-α, but elevated protein expressions of BMP-2, collagen II, and aggrecan. Naringin also increased disc matrix gene activity including aggrecan and Sox6, and decreased the gene expression of MMP3. CONCLUSION: Naringin effectively promotes the proliferation of degenerative human NP cells and improves the recuperation of the cells from degeneration by increasing expression of aggrecan, BMP-2, and Sox6 while inhibiting the expression of TNF-α and MMP3. This study suggests that naringin may represent an alternative therapeutic agent for disc degeneration.