Management of Hand and Wrist Injuries in NCAA Division I Football Players From a Single Institution: Factors Associated With Epidemiology, Surgical Intervention, and Return to Play.

Background: Upper extremity injuries account for approximately 16.9% of football injuries in the National Collegiate Athletic Association (NCAA). Purpose: To determine the epidemiology, management, and outcomes of hand/wrist injuries in collegiate football athletes so as to identify factors associated with surgical intervention and delayed return to play (RTP). Study Design: Descriptive epidemiology study. Methods: We retrospectively reviewed hand/wrist injuries that occurred within a single NCAA Division I football team from January 1, 2003, to December 31, 2020. Data analyzed included player position, college seniority, injury characteristics, injury management, surgical procedures performed, and timing of RTP. A univariate analysis was performed to identify factors associated with increased risk for surgical intervention and delayed (>21 days) RTP after hand and wrist injury in this cohort. Results: Overall, 124 patients with 168 hand/wrist injuries were identified (9.9 wrist/hand injuries per year). Sprain of the thumb metacarpophalangeal (MCP) joint ulnar collateral ligament (UCL) was the most common diagnosis (19.6%). Surgery was required in 22% of injuries, with injury of the UCL of the thumb MCP joint (8/37) being the most common indication. Injuries occurring during competitive games (odds ratio = 4.29; 95% CI, 1.2-15.9) were associated with an increased risk for surgery. Most (70%) injuries did not lead to time missed from football, whereas the remaining 30% resulted in an average of 33 ± 36 days missed. Conclusion: Over 17 athletic seasons, the annual incidence of hand and wrist injury in these NCAA Division I football players was 9.9 injuries per year, with 22% requiring surgical treatment. Injury to the UCL of the thumb MCP joint was the most common injury and indication for surgery, and 30% of injuries resulted in approximately 1 month lost. Injuries sustained in games were associated with operative management and delayed RTP.

Cutis Calcinosis of the Hand in 2 Patients With Symbrachydactyly.

Cutis calcinosis of the hand in the setting of symbrachydactyly is presented in 2 unique patients. Both lesions were treated based on the standard of care protocols with resection of the calcified mass and hand reconstruction, as appropriate. The patients healed uneventfully without recurrence of the calcification at a the 1-year follow-up. The association between symbrachydactyly and calcinosis cutis is discussed along with a hypothesis on the pathophysiologic mechanism that could potentially explain this unusual occurrence and why it might be more common than previously identified.

Free Vascularized Fibula Graft for Staged Reconstruction of Infected Humerus Nonunions.

OBJECTIVE: To evaluate the union rate and rate of postoperative complications in patients with septic nonunions of the humerus after a two-stage reconstruction using a free vascularized fibula graft. DESIGN: Retrospective case series. SETTING: Academic, tertiary referral center. PATIENTS/PARTICIPANTS: Adult patients with staged reconstruction for infected nonunion of the humerus with at least 2 years follow-up after vascularized fibula graft transfer. INTERVENTION: First, infected nonunion debridement with antibiotic spacer and external fixator placement. After antimicrobial treatment, free vascularized fibula transfer with internal fixation. MAIN OUTCOME MEASUREMENTS: Time to union, pain, affected extremity range of motion, and function. RESULTS: 10 patients with septic humerus nonunion treated with staged reconstruction using a free vascularized fibula graft, with a mean follow-up of 32.3 months were included. After the two-stage reconstruction using a free fibula, radiographic union was achieved in 6/10 patients, with a mean time to union of 19.9 weeks. The remaining 4 patients required an additional procedure with graft augmentation and/or implant revision. After the revision procedure, union was noted in 3/4 patients, 21 weeks postoperatively. Mean patient visual analog scale pain scores improved from 5.8 preoperatively to 0.9 at final follow-up ( P = 0.02). Postoperatively, mean elbow flexion was 110 ± 20 degrees and extension 15 ± 7.5 degrees. CONCLUSION: A two-stage reconstruction using a free fibula graft can be used in patients with septic nonunions of the humerus in the setting of multiple failed prior surgeries and compromised local biology. Additional procedures may be needed in some to achieve final union. LEVEL OF EVIDENCE: Therapeutic Level IV. See Instructions for Authors for a complete description of levels of evidence.

Influence of donor age and comorbidities on transduced human adipose-derived stem cell in vitro osteogenic potential.

Human adipose-derived mesenchymal stem cells (ASCs) transduced with a lentiviral vector system to express bone morphogenetic protein 2 (LV-BMP-2) have been shown to reliably heal bone defects in animal models. However, the influence of donor characteristics such as age, sex, race, and medical co-morbidities on ASC yield, growth and bone regenerative capacity, while critical to the successful clinical translation of stem cell-based therapies, are not well understood. Human ASCs isolated from the infrapatellar fat pads in 122 ASC donors were evaluated for cell growth characteristics; 44 underwent additional analyses to evaluate in vitro osteogenic potential, with and without LV-BMP-2 transduction. We found that while female donors demonstrated significantly higher cell yield and ASC growth rates, age, race, and the presence of co-morbid conditions were not associated with differences in proliferation. Donor demographics or the presence of comorbidities were not associated with differences in in vitro osteogenic potential or stem cell differentiation, except that transduced ASCs from healthy donors produced more BMP-2 at day 2. Overall, donor age, sex, race, and the presence of co-morbid conditions had a limited influence on cell yield, proliferation, self-renewal capacity, and osteogenic potential for non-transduced and transduced (LV-BMP-2) ASCs. These results suggest that ASCs are a promising resource for both autologous and allogeneic cell-based gene therapy applications.

Necrotizing Soft Tissue Infection of the Upper Extremity as a Manifestation of Hansen's Disease.

Hansen's disease is a well-described, largely historic infection that is caused by Mycobacterium leprae. Lucio's phenomenon is an aggressive, rare form of untreated lepromatous leprosy characterized by diffuse cutaneous lesions and systemic symptoms. To date, cases of necrotizing soft tissue infection in the setting of leprosy have rarely been reported in the literature. We present the case of a 51-year-old man with no known past medical history who presented for the evaluation of acute-on-chronic left upper extremity ulceration, soft tissue swelling, and pain. The patient was diagnosed with necrotizing soft tissue infection of the left upper extremity and underlying multibacillary lepromatous leprosy with Lucio's phenomenon. He underwent dermatofasciectomy of the affected extremity, followed by staged soft tissue coverage, including dermal allograft placement. Proper antibiotic management was also undertaken. In this article, we describe a case of previously undiagnosed leprosy with Lucio's phenomenon manifesting as necrotizing fasciitis of the upper extremity.

Clinical Efficacy of Bone Marrow Aspirate Concentrate Versus Stromal Vascular Fraction Injection in Patients With Knee Osteoarthritis: A Systematic Review and Meta-analysis.

BACKGROUND: Knee injection using either bone marrow aspirate concentrate (BMAC) or stromal vascular fraction (SVF) from adipose tissue has been shown to result in symptomatic improvement in patients with knee osteoarthritis (OA). It is still unclear whether one of these therapies is superior over the other. PURPOSE: To systematically report the clinical studies evaluating BMAC and SVF in the treatment of knee OA and to compare the clinical efficacy of these 2 injection therapies. STUDY DESIGN: Meta-analysis; Level of evidence, 4. METHODS: This meta-analysis was performed per the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) guidelines. Studies were included if they reported the clinical outcomes after a single BMAC or SVF injection in the knee joint of patients with OA. Studies evaluating preparations of culture-expanded stem cells were excluded. A random effects model was used; the clinical efficacy of BMAC or SVF injection was assessed using the standardized mean difference (SMD) and compared. Visual analog scale (VAS) scores for pain and Western Ontario and McMaster Universities Osteoarthritis (WOMAC) knee index were the primary outcomes. The level of statistical significance was set at P < .05. RESULTS: Ten studies and 472 patients with knee OA who received either BMAC (233 patients) or SVF (239 patients) were included. Patients who received an injection had improved VAS outcomes (mean ± SD): from 5.8 ± 1.3 to 2.6 ± 17 for BMAC and from 6.4 ± 1.4 to 3.4 ± 0.5 for SVF. They also experienced significantly reduced pain (SMD [VAS], 2.6 for BMAC and 3.4 for SVF) and improved function (SMD [WOMAC], 1.4 for BMAC and 1.2 for SVF). However, the SVF injection had a significantly greater effect on pain reduction than did the BMAC injection (P < .0001). Based on WOMAC, the clinical effect of BMAC versus SVF knee injection in patients with knee OA was equivalent (P = .626). Results were limited by the presence of publication bias as well as variability in the preparation methods utilized in the BMAC and SVF injection protocols. Complications were reported in 50% of the BMAC studies (knee stiffness, persistent knee swelling) and 67% of the SVF studies (knee swelling, knee pain, positive SVF cultures without symptoms of infection, and bleeding at the abdominal harvest site). CONCLUSION: A single BMAC or SVF injection into the knee joint of patients with OA resulted in symptomatic improvement at short-term follow-up. However, SVF seemed to be more effective than did BMAC in the reduction of knee pain. There was significant variation in the BMAC and SVF injection preparation techniques used across the studies and a lack of stratification of outcomes based on the radiologic classification of OA. Therefore, these results should be taken with caution.

Surgical management of humeral shaft nonunions. Success of a consistent protocol over 17 years.

INTRODUCTION: Surgical treatment of humeral shaft nonunions is characterized by variability of fixation methods, graft choices, and rates of union and iatrogenic radial nerve palsy. The aim of the current study is to evaluate the union rate of humeral shaft aseptic nonunions and the rate of postoperative complications following a consistent management protocol. PATIENTS AND METHODS: This is a retrospective review of 41 consecutive adult patients (23 female and 18 male with a mean age of 42 years) with aseptic nonunions of the humeral shaft treated by the senior author in our institution over a 17-year period. Nonunions were located in the middle third of the diaphysis in 33 patients, in the distal third in 6, and in the proximal third in 2 patients. Comorbidities were present in 49% of patients and the most common were smoking in 27% and diabetes mellitus in 17% of patients. Patients were treated at an average of 24 months after their injury. Surgical protocol consisted of careful dissection of the radial nerve, debridement of the nonunion site, stable plate fixation and augmentation of local biology. RESULTS: Thirty-eight patients had mean clinical and radiographic follow-up of 9.4 months. All 38 nonunions healed at a mean time of 3.5 months. There were no persistent nonunions and no failures of fixation. None of the 40 patients with an intact radial nerve preoperatively developed any signs of radial nerve compromise after surgery. Complications consisted of one superficial infection (2%) that resolved with oral antibiotics and one deep infection (2%) that required implant removal and debridement. The mean pain score on the visual analog scale was 0.7. Mean elbow range of motion was 125 degrees with a mean extension deficit of 5 degrees and mean flexion of 130 degrees. CONCLUSIONS: Our surgical protocol achieved consistent healing of nonunions of the humeral shaft with a low complication rate and no iatrogenic radial nerve palsy, even in long-standing nonunions in patients with comorbidities.

Regional Gene Therapy with Transduced Human Cells: The Influence of "Cell Dose" on Bone Repair.

Regional gene therapy using a lentiviral vector containing the BMP-2 complementary DNA (cDNA) has been shown to heal critical-sized bone defects in rodent models. An appropriate "cellular dose" needs to be defined for eventual translation into human trials. The purpose of this study was to evaluate bone defect healing potential and quality using three different doses of transduced human bone marrow cells (HBMCs). HBMCs were transduced with a lentiviral vector containing either BMP-2 or green fluorescent protein (GFP). All cells were loaded onto compression-resistant matrices and implanted in the bone defect of athymic rats. Treatment groups included femoral defects that were treated with a low-dose (1 × 106 cells), standard-dose (5 × 106 cells), and high-dose (1.5 × 107 cells) HBMCs transduced with lentiviral vector containing BMP-2 cDNA. The three control groups were bone defects treated with HBMCs that were either nontransduced or transduced with vector containing GFP. All animals were sacrificed at 12 weeks. The bone formed in each defect was evaluated with plain radiographs, microcomputed tomography (microCT), histomorphometric analysis, and biomechanical testing. Bone defects treated with higher doses of BMP-2-producing cells were more likely to have healed (6/14 of the low-dose group; 12/14 of the standard-dose group; 14/14 of the high-dose group; χ2(2) = 15.501, p < 0.001). None of the bone defects in the control groups had healed. Bone defects treated with high dose and standard dose of BMP-2-producing cells consistently outperformed those treated with a low dose in terms of bone formation, as assessed by microCT and histomorphometry, and biomechanical parameters. However, statistical significance was only seen between defects treated with high dose and low dose. Larger doses of BMP-2-producing cells were associated with a higher likelihood of forming heterotopic ossification. Femurs treated with a standard- and high-dose BMP-2-producing cells demonstrated similar healing and biomechanical properties. Increased doses of BMP-2 delivered through higher cell doses have the potential to heal large bone defects. Adapting regional gene therapy for use in humans will require a balance between promoting bone repair and limiting heterotopic ossification. Impact statement Critical bone loss may result from complex traumatic bone injury (i.e., open fracture or blast injury), revision total joint arthroplasty, and spine pseudoarthrosis. This is a challenging clinical problem to treat and regional gene therapy is an innovative means of addressing it. This study provides information regarding the quantity of cells or "cell dose" of transduced cells needed to treat a critical-sized bone defect in a rat model. This information may be extrapolated for use in humans in future trials.

Limited potential of AAV-mediated gene therapy in transducing human mesenchymal stem cells for bone repair applications.

Adeno-associated viral vectors (AAV) are unique in their ability to transduce a variety of both dividing and nondividing cells, with significantly lower risk of random genomic integration and with no known pathogenicity in humans, but their role in ex vivo regional gene therapy for bone repair has not been definitively established. The goal of this study was to test the ability of AAV vectors carrying the cDNA for BMP-2 to transduce human mesenchymal stem cells (MSCs), produce BMP-2, and induce osteogenesis in vitro as compared with lentiviral gene therapy with a two-step transcriptional amplification system lentiviral vector (LV-TSTA). To this end, we created two AAV vectors (serotypes 2 and 6) expressing the target transgene; eGFP or BMP-2. Transduction of human MSCs isolated from bone marrow (BMSCs) or adipose tissue (ASCs) with AAV2-eGFP and AAV6-eGFP led to low transduction efficiency (BMSCs: 3.57% and 8.82%, respectively, ASCs: 6.17 and 20.2%, respectively) and mean fluorescence intensity as seen with FACS analysis 7 days following transduction, even at MOIs as high as 106. In contrast, strong eGFP expression was detectable in all of the cell types post transduction with LV-TSTA-eGFP. Transduction with BMP-2 producing vectors led to minimal BMP-2 production in AAV-transduced cells 2 and 7 days following transduction. In addition, transduction of ASCs and BMSCs with AAV2-BMP-2 and AAV6-BMP-2 did not enhance their osteogenic potential as seen with an alizarin red assay. In contrast, the LV-TSTA-BMP-2-transduced cells were characterized by an abundant BMP-2 production and induction of the osteogenic phenotype in vitro (p < 0.001 vs. AAV2 and 6). Our results demonstrate that the AAV2 and AAV6 vectors cannot induce a significant transgene expression in human BMSCs and ASCs, even at MOIs as high as 106. The LV-TSTA vector is significantly superior in transducing human MSCs; thus this vector would be preferable when developing an ex vivo regional gene therapy strategy for clinical use in orthopedic surgery applications.

Ballistic trauma patients have decreased early narcotic demand relative to blunt trauma patients: Blunt ballistic injury opioid use.

OBJECTIVES: Blunt and ballistic injuries are two common injury mechanisms encountered by orthopaedic traumatologists. However the intrinsic nature of these injures may necessitate differences in operative and post-operative care. Given the evolving opioid crisis in the medical community, considerable attention has been given to appropriate management of pain; particularly in orthopaedic patients. We sought to evaluate relative postoperative narcotic use in blunt injuries and ballistic injuries. DESIGN: Retrospective Cohort Study. SETTING: Academic Level-1 Trauma Center. PATIENTS: 96 Patients with blunt or ballistic fractures. INTERVENTION: Inpatient narcotic pain management after orthopaedic fracture management. MAIN OUTCOME MEASUREMENTS: Morphine equivalent units (MEU). RESULTS: Patients with blunt injuries had a higher MEU compared to ballistic injuries in the first 24 hours postoperatively (35.0 vs 29.5 MEU, p=0.02). There were no differences in opiate consumption 24-48 hours (34.8 vs 28.0 MEU), 48 hours - 7 days post op (28.4 vs 30.4 MEU) or the 24 hours before discharge (30.0 vs 28.6 MEU). On multivariate analysis, during the 24-48 hours and 24 hours before discharge timepoints total EBL was associated with increased opioid usage. During days 3-7 (p<0.001) and in the final 24 hours prior to discharge (p=0.012), the number of orthopaedic procedures was a predictor of opioid consumption. CONCLUSION: Blunt injuries required an increased postoperative narcotic consumption during the first 24 hours of inpatient stay following orthopedic fracture fixation. However, there was no difference at other time points. Immediate post-operative pain regimens may be decreased for patients with ballistic injuries. LEVEL OF EVIDENCE: III.

Ex vivo regional gene therapy with human adipose-derived stem cells for bone repair.

BACKGROUND: The treatment of complex bone loss scenarios remains challenging. This study evaluates the efficacy of ex vivo regional gene therapy using transduced human adipose-derived stem cells (ASCs) overexpressing bone morphogenetic protein-2 (BMP-2) to treat critical-sized bone defects. METHODS: Critical-sized femoral defects created surgically in immunocompromised rats were treated with ASCs transduced with a lentivirus encoding BMP-2 (Group 1, n = 14), or green fluorescent protein (Group 2, n = 5), nontransduced ASCs (Group 3, n = 5), or rhBMP-2 (Group 4, n = 14). At 12 weeks, femurs were evaluated for quantity and quality of bone formation with plain radiographs, micro-computed tomography, histology/histomorphometry, and biomechanical strength testing. RESULTS: Thirteen of 14 samples in Group 1 and all 14 samples in Group 4 showed radiographic healing, while no samples in either Groups 2 or 3 healed. Groups 1 and 4 had significantly higher radiographic scores (p < 0.001), bone volume fraction (BV/TV) (p < 0.001), and bone area fraction (BA/TA) than Groups 2 and 3 (p < 0.001). Radiographic scores, BV/TV, and BA/TA were not significantly different between Groups 1 and 4. No difference with regards to mean torque, rotation at failure, torsional stiffness, and energy to failure was seen between Groups 1 and 4. CONCLUSIONS: Human ASCs modified to overexpress BMP-2 resulted in abundant bone formation, with the quality of bone comparable to that of rhBMP-2. This strategy represents a promising approach in the treatment of large bone defects in the clinical setting. CLINICAL RELEVANCE: Large bone defects may require sustained protein production to induce an appropriate osteoinductive response. Ex vivo regional gene therapy using a lentiviral vector has the potential to be part of a comprehensive tissue engineering strategy for treating osseous defects.

Functional outcomes of tendon transfer for brachial plexus birth palsy using the Hoffer technique.

AIMS: To describe and analyze the mid-term functional outcomes of a large series of patients who underwent the Hoffer procedure for brachial plexus birth palsy (BPBP). METHODS: All patients who underwent the Hoffer procedure with minimum two-year follow-up were retrospectively reviewed. Active shoulder range of movement (ROM), aggregate modified Mallet classification scores, Hospital for Sick Children Active Movement Scale (AMS) scores, and/or Toronto Test Scores were used to assess functional outcomes. Subgroup analysis based on age and level of injury was performed. Risk factors for subsequent humeral derotational osteotomy and other complications were also assessed. A total of 107 patients, average age 3.9 years (1.6 to 13) and 59% female, were included in the study with mean 68 months (24 to 194) follow-up. RESULTS: All patients demonstrated statistically significant improvement in all functional outcomes and active shoulder abduction and external rotation ROM (p < 0.001). Patients < 2.5 years of age had higher postoperative AMS, abduction ROM and strength scores, and aggregate postoperative Toronto scores (p ≤ 0.035) compared to patients ≥ 2.5 years old. There were 17 patients (16%) who required a subsequent humeral derotational osteotomy; lower preoperative AMS external rotation scores and external rotation ROM were predictive risk factors (p ≤ 0.016). CONCLUSION: Patients with BPBP who underwent the Hoffer procedure demonstrated significant improvement in postoperative ROM, strength, and functional outcome scores at mid-term follow-up. Patients younger than 2.5 years at the time of surgery generally had better functional outcomes. Limited preoperative external rotation strength and ROM were significantly associated with requirement for subsequent humeral derotational osteotomy. In our chort significant improvements in shoulder function were obtained after the Hoffer procedure for BPBP. Cite this article: Bone Joint J 2020;102-B(2):246-253.

Intraoperative Subluxation of the Ulnar Nerve: Use of a Triceps Sling Reconstruction Technique.

PURPOSE: To determine whether the triceps sling reconstruction technique is a safe and effective treatment of intraoperative ulnar nerve subluxation after in situ decompression. METHODS: Twelve patients who underwent a triceps sling reconstruction for intraoperative ulnar nerve subluxation after in situ release were retrospectively reviewed. The triceps sling technique consists of harvesting a small, distally based strip of triceps tendon and suturing the proximal end of the strip to the posterior aspect of the released Osborne ligament. Thus, a sling is created between the medial epicondyle and the olecranon, preventing the nerve from subluxating. Patients were clinically evaluated before and after surgery. Visual analog scale pain scores, static 2-point discrimination, strength, and Disabilities of the Arm, Shoulder, and Hand score were assessed. RESULTS: At a mean follow-up of 31 months (range, 24-38 months), there was a significant improvement in mean visual analog pain scores from 8.6 to 0.2. Static 2-point discrimination was improved from a mean of 9.1 mm before surgery to 5.7 mm afterward. Strength improved by a mean of 33% and 30% with grip and pinch, respectively. Mean Disabilities of the Arm, Shoulder, and Hand score improved from 45.9 to 3.7. No subluxation of the ulnar nerve was noted after surgery. No other complications were noted. No reoperations were required during the follow-up period. CONCLUSIONS: Triceps sling reconstruction is a safe treatment in patients with intraoperative ulnar nerve subluxation after in situ decompression. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

Ex vivo gene therapy using human bone marrow cells overexpressing BMP-2: "Next-day" gene therapy versus standard "two-step" approach.

Traditionally, ex vivo gene therapy involves a two-step approach, with culture expansion of cells prior to transduction and implantation. We have tried to simplify this strategy and eliminate the time and cost associated with culture expansion, by introducing "next-day" regional gene therapy using human bone marrow cells. The purpose of this study was to determine whether a lentiviral vector (LV) carrying the cDNA for BMP-2 can transduce freshly isolated human BM cells, leading to abundant BMP production and bone formation in vivo, and evaluate the in vivo osteoinductive potential of "next-day" gene therapy and the standard "two-step" tissue culture expansion approach. To this end, human bone marrow cells (HBMC) from patients undergoing total hip arthroplasty were harvested, transduced with a BMP-2-expressing LV either overnight ("next day" gene therapy; ND) or after culture expansion (cultured "two-step" approach; C) and then implanted into a rat critical-sized femoral defect. The animals were randomly assigned to one of the following groups: I; ND-HBMC transduced with LV-TSTA BMP-2, II; ND-HBMC transduced with LV-TSTA GFP, III; non-transduced ND-HBMC; IV; C-HBMC transduced with LV-TSTA BMP-2, V; C-HBMC transduced with LV-TSTA-GFP, VI; non-transduced C-HBMC. Treatment with either "next-day" or cultured HBMC demonstrated a significant increase in new bone formation compared with all negative control groups as seen in plain radiographs, microCT and histologic/histomorphometric analysis. At 12 weeks post-op, complete defect union on plain X-rays occurred in 7/14 animals in the ND-HBMC/BMP-2 group and 12/14 in the C-HBMC/BMP-2 treated rats. The two-step approach was associated with more consistent results, a higher union rate, and superiority with regards to all of the studied bone healing parameters. In this study we demonstrate proof of concept that BMP-2-transduced human bone marrow cells can be used to enhance bone healing in segmental bone defects, and that regional gene therapy using lentiviral transduction has the osteoinductive potential to heal large bone defects in clinical settings.

Regional gene therapy with 3D printed scaffolds to heal critical sized bone defects in a rat model.

The objective of the present study was to assess the ability of transduced rat bone marrow cells (RBMCs) that overexpress BMP-2 loaded on a three-dimensionally (3D) printed scaffold to heal a critical sized rat femoral defect. Tricalcium phosphate (TCP) scaffolds were 3D printed to fit a critical sized rat femoral defect. The RBMCs were transduced with a lentiviral (LV) vector expressing BMP-2 or GFP. The rats were randomized into the following treatment groups: (1) RBMC/LV-BMP-2 + TCP, (2) RBMC/LV-GFP + TCP, (3) nontransduced RBMCs + TCP, (4) TCP scaffold alone. The animals were euthanized at 12 weeks and evaluated with plain radiographs, microcomputed tomography (micro-CT), histology, histomorphometry, and biomechanically. Each LV-BMP-2 + TCP treated specimen demonstrated complete healing of the femoral defect on plain radiographs and micro-CT. No femurs healed in the control groups. Micro-CT demonstrated that LV-BMP-2 + TCP treated femoral defects formed 197% more bone volume compared to control groups (p < 0.05). Histologic analysis demonstrated bone formation across the TCP scaffold, uniting the femoral defect on both ends in the LV-BMP-2 + TCP treated specimens. Biomechanical assessment demonstrated similar stiffness (p = 0.863), but lower total energy to failure, peak torque, and peak displacement (p < 0.001) of the femurs treated with LV-BMP-2 + TCP when compared to the contralateral control femur. Regional gene therapy induced overexpression of BMP-2 via transduced RBMCs combined with an osteoconductive 3D printed TCP scaffold can heal a critically sized femoral defect in an animal model. The combination of regional gene therapy and 3D printed osteoconductive scaffolds has significant clinical potential to enhance bone regeneration.

Regulated ex vivo regional gene therapy for bone repair using an inducible caspase-9 suicide gene system.

In order to adapt ex vivo regional gene therapy for clinical applications in orthopaedic surgery, safety issues must be considered. In this study we developed a suicide approach using a dual gene expression two step transcriptional amplification lentiviral vector (LV-TSTA) encoding BMP-2 and an inducible caspase 9 (iC9) system that selectively induces apoptosis upon activation with a chemical inducer of dimerization (CID). Transduction of rat bone marrow stromal cells (RBMSCs) with LV-TSTA-iC9/BMP-2 led to abundant BMP-2 production (90.3 ± 7.9 ng/24 h/106 cells) in vitro and stimulated bone formation in a mouse muscle pouch in the absence of CID. Moreover it was shown that CID could be used to selectively induce apoptosis in iC9-transduced cells both in vitro and in vivo. Double exposure to serial dilutions of CID decreased in vitro production of BMP-2 by 85-87% and Luc activity by 97-99% in iC9/BMP-2 or iC9/Luc-transduced cells respectively. Early administration of CID (Days 0-1 post-op) in mice implanted with iC9/BMP-2-transduced RBMSCs was effective in blocking bone formation, indicating that CID was toxic to the transduced cells. In iC9/Luc-implanted mice, late administration of two doses of CID (Days 27-28 post-op) significantly reduced the luciferase signal. The current study provides proof of concept for the potential clinical application of regulated gene therapy to promote bone repair.

Lentiviral Gene Therapy for Bone Repair Using Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells.

Umbilical cord blood (UCB) has been increasingly explored as an alternative source of stem cells for use in regenerative medicine due to several advantages over other stem-cell sources, including the need for less stringent human leukocyte antigen matching. Combined with an osteoinductive signal, UCB-derived mesenchymal stem cells (MSCs) could revolutionize the treatment of challenging bone defects. This study aimed to develop an ex vivo regional gene-therapy strategy using BMP-2-transduced allogeneic UCB-MSCs to promote bone repair. To this end, human UCB-MSCs were transduced with a lentiviral vector carrying the cDNA for BMP-2 (LV-BMP-2). In vitro assays to determine the UCB-MSC osteogenic potential and BMP-2 production were followed by in vivo implantation of LV-BMP-2-transduced UCB-MSCs in a mouse hind-limb muscle pouch. Non-transduced and LV-GFP-transduced UCB-MSCs were used as controls. Transduction with LV-BMP-2 was associated with abundant BMP-2 production and induction of osteogenic differentiation in vitro. Implantation of BMP-2-transduced UCB-MSCs led to robust heterotopic bone formation 4 weeks postoperatively, as seen on radiographs and histology. These results, along with the fact that UCB-MSCs can be easily collected with no donor-site morbidity and low immunogenicity, suggest that UCB might be a preferable allogeneic source of MSCs to develop an ex vivo gene-therapy approach to treat difficult bone-repair scenarios.

Failure in diagnosis and under-treatment of osteoporosis in elderly patients with fragility fractures.

We evaluated whether osteoporosis is adequately managed and treated in patients suffering from fragility fractures. Factors that influenced osteoporosis diagnosis and treatment rates were also assessed. To this end, patients with the principal diagnosis of low-energy hip, vertebral, or distal radius fractures were recruited for the study. Collected data included risk factors for osteoporosis, history of previous fractures, known history of osteoporosis, and osteoporosis treatment at the time of admission. The patients' prefracture risk profile was also assessed to determine whether osteoporosis could have been identified prior to the index fracture. We identified 308 patients with fragility fractures, including 214 hip, 41 vertebral, and 53 distal radius fractures. Overall, 238 patients (77.3%) had at least one risk factor for osteoporosis. Eighty-eight patients (28.6%) had sustained ≥ 1 prior fragility fractures in the past. However, only 79 patients (25.6%) were aware that they had osteoporosis and even fewer (66 patients, 21.4%) had been receiving osteoporosis treatment preceding the current admission. Anti-osteoporotic agents were more commonly prescribed in patients 66-75 years old (p = 0.008), with a family history of osteoporosis (p = 0.009) or history of a prior fragility fracture (p = 0.012). The treatment rate was higher in women than men (p = 0.026) and in patients with vertebral or multiple prior fractures compared to patients with prior hip fractures. The current study provides evidence that individuals who experience fragility fractures are not adequately managed for osteoporosis. Only few of the historically known risk factors for osteoporosis were adequately recognized and associated with osteoporosis evaluation and treatment.

Cryopreservation of Human Adipose-Derived Stem Cells for Use in Ex Vivo Regional Gene Therapy for Bone Repair.

The development of an ex vivo regional gene therapy clinical pathway using adipose-derived stem cells (ASCs) may require cryopreservation for cell culture, storage, and transport prior to clinical use. ASCs isolated from five donors were transduced with a lentiviral vector containing BMP-2. Three groups were assessed: transduction without cell freezing (group 1), freezing of cells for 3 weeks followed by transduction (group 2), and cell transduction prior to freezing (group 3). Nontransduced cells were used as a control. The cluster of differentiation (CD) marker profiles, cell number, BMP-2 production, and osteogenic potential were measured. The CD marker profile (CD44, CD73, CD90, and CD105) was unchanged after cryopreservation. Cell number was equivalent among cryopreservation protocols in transduced and nontransduced cells. There was a trend toward decreased BMP-2 production in group 3 compared to groups 1 and 2. Osteogenic potential based on Alizarin red concentration was higher in group 2 compared to group 3, with no difference compared to group 1. Freezing ASCs prior to transduction with a lentiviral vector containing BMP-2 has no detrimental effect on cell number, BMP-2 production, osteogenic potential, or immunophenotype. Transduction prior to freezing, however, may limit the BMP-2 production and potential osteogenic differentiation of the ASCs.

In vitro evaluation of a lentiviral two-step transcriptional amplification system using GAL4FF transactivator for gene therapy applications in bone repair.

In this study, we developed a lentiviral two-step transcriptional amplification (TSTA) system expressing bone morphogenetic protein-2 (BMP-2) under the control of a GAL4FF transactivator to enhance gene expression and limit toxicity for bone repair applications. To this end human MSCs, isolated from bone marrow or adipose tissue, were transduced overnight with a LV-TSTA system (GAL4FF or GAL4vp16) expressing BMP-2 or GFP and evaluated in vitro for transduction efficiency, mean fluorescence intensity, cell viability, and BMP-2 production. FACS analysis of GFP-transduced MSCs confirmed successful transduction with the GAL4FF+GFP vector. Moreover, ELISA demonstrated abundant BMP-2 production by GAL4FF+BMP2-transduced human MSCs over a period of 8 weeks, with minimal cytotoxicity at all time points. Compared to GAL4vp16, GAL4FF was superior with respect to BMP production at 1, 2, 4, 6, and 8 weeks in BMSCs. In ASCs, GAL4FF was still associated with higher BMP-2 production at weeks 2-8, but this difference was not as prominent as in BMSCs. To our knowledge, this is the first report of GAL4FF-mediated BMP-2 production by human BMSCs and ASCs. Compared to the standard GAL4vp16TSTA vector, GAL4FF was associated with lower cytotoxicity and higher in vitro gene expression in both BMSCs and ASCs.