Assessing the Resident Progenitor Cell Population and the Vascularity of the Adult Human Meniscus.

PURPOSE: To identify, characterize, and compare the resident progenitor cell populations within the red-red, red-white, and white-white (WW) zones of freshly harvested human cadaver menisci and to characterize the vascularity of human menisci using immunofluorescence and 3-dimensional (3D) imaging. METHODS: Fresh adult human menisci were harvested from healthy donors. Menisci were enzymatically digested, mononuclear cells isolated, and characterized using flow cytometry with antibodies against mesenchymal stem cell surface markers (CD105, CD90, CD44, and CD29). Cells were expanded in culture, characterized, and compared with bone marrow-derived mesenchymal stem cells. Trilineage differentiation potential of cultured cells was determined. Vasculature of menisci was mapped in 3D using a modified uDisco clearing and immunofluorescence against vascular markers CD31, lectin, and alpha smooth muscle actin. RESULTS: There were no significant differences in the clonogenicity of isolated cells between the 3 zones. Flow cytometry showed presence of CD44+CD105+CD29+CD90+ cells in all 3 zones with high prevalence in the WW zone. Progenitors from all zones were found to be potent to differentiate to mesenchymal lineages. Larger vessels in the red-red zone of meniscus were observed spanning toward red-white, sprouting to smaller arterioles and venules. CD31+ cells were identified in all zones using the 3D imaging and co-localization of additional markers of vasculature (lectin and alpha smooth muscle actin) was observed. CONCLUSIONS: The presence of resident mesenchymal progenitors was evident in all 3 meniscal zones of healthy adult donors without injury. In addition, our results demonstrate the presence of vascularization in the WW zone. CLINICAL RELEVANCE: The existence of progenitors and presence of microvasculature in the WW zone of the meniscus suggests the potential for repair and biologic augmentation strategies in that zone of the meniscus in young healthy adults. Further research is necessary to fully define the functionality of the meniscal blood supply and its implications for repair.

Advanced Glycation End Product Inhibitor Pyridoxamine Attenuates IVD Degeneration in Type 2 Diabetic Rats.

Type 2 diabetes mellitus (T2DM) is associated with advanced glycation end product (AGE) enrichment and considered a risk factor for intervertebral disc (IVD) degeneration. We hypothesized that systemic AGE inhibition, achieved using pyridoxamine (PM), attenuates IVD degeneration in T2DM rats. To induce IVD degeneration, lumbar disc injury or sham surgery was performed on Zucker Diabetic Sprague Dawley (ZDSD) or control Sprague Dawley (SD) rats. Post-surgery, IVD-injured ZDSD rats received daily PM dissolved in drinking water or water only. The resulting groups were SD uninjured, SD injured, ZDSD uninjured, ZDSD injured, and ZDSD injured + PM. Levels of blood glycation and disc degeneration were investigated. At week 8 post-surgery, glycated serum protein (GSP) levels were increased in ZDSDs compared to SDs. PM treatment attenuated this increase. Micro-MRI analysis demonstrated IVD dehydration in injured versus uninjured SDs and ZDSDs. In the ZDSD injured + PM group, IVD dehydration was diminished compared to ZDSD injured. AGE levels were decreased and aggrecan levels increased in ZDSD injured + PM versus ZDSD injured rats. Histological and immunohistochemical analyses further supported the beneficial effect of PM. In summary, PM attenuated GSP levels and IVD degeneration processes in ZDSD rats, demonstrating its potential to attenuate IVD degeneration in addition to managing glycemia in T2DM.

Ultrasound-Mediated Gene Delivery Enhances Tendon Allograft Integration in Mini-Pig Ligament Reconstruction.

Ligament injuries occur frequently, substantially hindering routine daily activities and sports participation in patients. Surgical reconstruction using autogenous or allogeneic tissues is the gold standard treatment for ligament injuries. Although surgeons routinely perform ligament reconstructions, the integrity of these reconstructions largely depends on adequate biological healing of the interface between the ligament graft and the bone. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would lead to significantly improved ligament graft integration. To test this hypothesis, an anterior cruciate ligament reconstruction procedure was performed in Yucatan mini-pigs. A collagen scaffold was implanted in the reconstruction sites to facilitate recruitment of endogenous mesenchymal stem cells. Ultrasound-mediated reporter gene delivery successfully transfected 40% of cells recruited to the reconstruction sites. When BMP-6 encoding DNA was delivered, BMP-6 expression in the reconstruction sites was significantly enhanced. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to significantly enhanced osteointegration in all animals 8 weeks after surgery. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively improve ligament reconstruction in large animals, thereby addressing a major unmet orthopedic need and offering new possibilities for translation to the clinical setting.

PTH Induces Systemically Administered Mesenchymal Stem Cells to Migrate to and Regenerate Spine Injuries.

Osteoporosis affects more than 200 million people worldwide leading to more than 2 million fractures in the United States alone. Unfortunately, surgical treatment is limited in patients with low bone mass. Parathyroid hormone (PTH) was shown to induce fracture repair in animals by activating mesenchymal stem cells (MSCs). However, it would be less effective in patients with fewer and/or dysfunctional MSCs due to aging and comorbidities. To address this, we evaluated the efficacy of combination i.v. MSC and PTH therapy versus monotherapy and untreated controls, in a rat model of osteoporotic vertebral bone defects. The results demonstrated that combination therapy significantly increased new bone formation versus monotherapies and no treatment by 2 weeks (P < 0.05). Mechanistically, we found that PTH significantly enhanced MSC migration to the lumbar region, where the MSCs differentiated into bone-forming cells. Finally, we used allogeneic porcine MSCs and observed similar findings in a clinically relevant minipig model of vertebral defects. Collectively, these results demonstrate that in addition to its anabolic effects, PTH functions as an adjuvant to i.v. MSC therapy by enhancing migration to heal bone loss. This systemic approach could be attractive for various fragility fractures, especially using allogeneic cells that do not require invasive tissue harvest.

Quantitative chemical exchange saturation transfer MRI of intervertebral disc in a porcine model.

PURPOSE: Previous studies have associated low pH in intervertebral discs (IVDs) with discogenic back pain. The purpose of this study was to determine whether quantitative CEST (qCEST) MRI can be used to detect pH changes in IVDs in vivo. METHODS: The exchange rate ksw between glycosaminoglycan (GAG) protons and water protons was determined from qCEST analysis. Its dependence on pH value was investigated in GAG phantoms with varying pH and concentrations. The relationship between ksw and pH was studied further in vivo in a porcine model on a 3T MR scanner and validated using a pH meter. Sodium lactate was injected into the IVDs to induce various pH values within the discs ranging from 5 to 7. RESULTS: Phantom and animal results revealed that ksw measured using qCEST MRI is highly correlated with pH level. In the animal studies, the relationship can be described as ksw =9.2 × 106 × 10-pH + 196.9, R2 = 0.7883. CONCLUSION: The exchange rate between GAG and water protons determined from qCEST MRI is closely correlated with pH value. This technique has the potential to noninvasively measure pH in the IVDs of patients with discogenic pain. Magn Reson Med 76:1677-1683, 2016. © 2016 International Society for Magnetic Resonance in Medicine.

Detection of low back pain using pH level-dependent imaging of the intervertebral disc using the ratio of R1ρ dispersion and -OH chemical exchange saturation transfer (RROC).

PURPOSE: Low pH is associated with intervertebral disc (IVD)-generated low back pain (LBP). The purpose of this work was to develop an in vivo pH level-dependent magnetic resonance imaging (MRI) method for detecting discogenic LBP, without using exogenous contrast agents. METHODS: The ratio of R1ρ dispersion and chemical exchange saturation transfer (CEST) (RROC) was used for pH-level dependent imaging of the IVD while eliminating the effect of labile proton concentration. The technique was validated by numerical simulations and studies on phantoms and ex vivo porcine spines. Four male (ages 42.8 ± 18.3) and two female patients (ages 55.5 ± 2.1) with LBP and scheduled for discography were examined with the method on a 3.0 Tesla MR scanner. RROC measurements were compared with discography outcomes using paired t-test. RESULTS: Simulation and phantom results indicated RROC is a concentration independent and pH level-dependent technique. Porcine spine study results found higher RROC value was related to lower pH level. Painful discs based on discography had significant higher RROC values than those with negative diagnosis (P < 0.05). CONCLUSION: RROC imaging is a promising pH level dependent MRI technique that has the potential to be a noninvasive imaging tool to detect painful IVDs in vivo.

Reliable chemical exchange saturation transfer imaging of human lumbar intervertebral discs using reduced-field-of-view turbo spin echo at 3.0 T.

The reduced field-of-view (rFOV) turbo-spin-echo (TSE) technique, which effectively suppresses bowel movement artifacts, is developed for the purpose of chemical exchange saturation transfer (CEST) imaging of the intervertebral disc (IVD) in vivo. Attempts to quantify IVD CEST signals in a clinical setting require high reliability and accuracy, which is often compromised in the conventionally used technique. The proposed rFOV TSE CEST method demonstrated significantly superior reproducibility when compared with the conventional technique on healthy volunteers, implying it is a more reliable measurement. Phantom study revealed a linear relation between CEST signal and glycosaminoglycan (GAG) concentration. The feasibility of detecting IVD degeneration was demonstrated on a healthy volunteer, indicating that the proposed method is a promising tool to quantify disc degeneration.

PTH promotes allograft integration in a calvarial bone defect.

Allografts may be useful in craniofacial bone repair, although they often fail to integrate with the host bone. We hypothesized that intermittent administration of parathyroid hormone (PTH) would enhance mesenchymal stem cell recruitment and differentiation, resulting in allograft osseointegration in cranial membranous bones. Calvarial bone defects were created in transgenic mice, in which luciferase is expressed under the control of the osteocalcin promoter. The mice were given implants of allografts with or without daily PTH treatment. Bioluminescence imaging (BLI) was performed to monitor host osteprogenitor differentiation at the implantation site. Bone formation was evaluated with the aid of fluorescence imaging (FLI) and microcomputed tomography (µCT) as well as histological analyses. Reverse transcription polymerase chain reaction (RT-PCR) was performed to evaluate the expression of key osteogenic and angiogenic genes. Osteoprogenitor differentiation, as detected by BLI, in mice treated with an allograft implant and PTH was over 2-fold higher than those in mice treated with an allograft implant without PTH. FLI also demonstrated that the bone mineralization process in PTH-treated allografts was significantly higher than that in untreated allografts. The µCT scans revealed a significant increase in bone formation in allograft + PTH treated mice comparing to allograft + PBS treated mice. The osteogenic genes osteocalcin (Oc/Bglap) and integrin binding sialoprotein (Ibsp) were upregulated in the allograft + PTH treated animals. In summary, PTH treatment enhances osteoprogenitor differentiation and augments bone formation around structural allografts. The precise mechanism is not clear, but we show that infiltration pattern of mast cells, associated with the formation of fibrotic tissue, in the defect site is significantly affected by the PTH treatment.

Retention of Human iPSC-Derived or Primary Cells Following Xenotransplantation into Rat Immune-Privileged Sites.

In regenerative medicine, experimental animal models are commonly used to study potential effects of human cells as therapeutic candidates. Although some studies describe certain cells, such as mesenchymal stromal cells (MSC) or human primary cells, as hypoimmunogenic and therefore unable to trigger strong inflammatory host responses, other studies report antibody formation and immune rejection following xenotransplantation. Accordingly, the goal of our study was to test the cellular retention and survival of human-induced pluripotent stem cell (iPSCs)-derived MSCs (iMSCs) and primary nucleus pulposus cells (NPCs) following their xenotransplantation into immune-privileged knee joints (14 days) and intervertebral discs (IVD; 7 days) of immunocompromised Nude and immunocompetent Sprague Dawley (SD) rats. At the end of both experiments, we could demonstrate that both rat types revealed comparably low levels of systemic IL-6 and IgM inflammation markers, as assessed via ELISA. Furthermore, the number of recovered cells was with no significant difference between both rat types. Conclusively, our results show that xenogeneic injection of human iMSC and NPC into immunoprivileged knee and IVD sites did not lead to an elevated inflammatory response in immunocompetent rats when compared to immunocompromised rats. Hence, immunocompetent rats represent suitable animals for xenotransplantation studies targeting immunoprivileged sites.

iPSC-derived tenocytes seeded on microgrooved 3D printed scaffolds for Achilles tendon regeneration.

Tendons and ligaments have a poor innate healing capacity, yet account for 50% of musculoskeletal injuries in the United States. Full structure and function restoration postinjury remains an unmet clinical need. This study aimed to assess the application of novel three dimensional (3D) printed scaffolds and induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) overexpressing the transcription factor Scleraxis (SCX, iMSCSCX+ ) as a new strategy for tendon defect repair. The polycaprolactone (PCL) scaffolds were fabricated by extrusion through a patterned nozzle or conventional round nozzle. Scaffolds were seeded with iMSCSCX+ and outcomes were assessed in vitro via gene expression analysis and immunofluorescence. In vivo, rat Achilles tendon defects were repaired with iMSCSCX+ -seeded microgrooved scaffolds, microgrooved scaffolds only, or suture only and assessed via gait, gene expression, biomechanical testing, histology, and immunofluorescence. iMSCSCX+ -seeded on microgrooved scaffolds showed upregulation of tendon markers and increased organization and linearity of cells compared to non-patterned scaffolds in vitro. In vivo gait analysis showed improvement in the Scaffold + iMSCSCX+ -treated group compared to the controls. Tensile testing of the tendons demonstrated improved biomechanical properties of the Scaffold + iMSCSCX+ group compared with the controls. Histology and immunofluorescence demonstrated more regular tissue formation in the Scaffold + iMSCSCX+ group. This study demonstrates the potential of 3D-printed scaffolds with cell-instructive surface topography seeded with iMSCSCX+ as an approach to tendon defect repair. Further studies of cell-scaffold constructs can potentially revolutionize tendon reconstruction by advancing the application of 3D printing-based technologies toward patient-specific therapies that improve healing and functional outcomes at both the cellular and tissue level.

Intervertebral disc human nucleus pulposus cells associated with back pain trigger neurite outgrowth in vitro and pain behaviors in rats.

Low back pain (LBP) is often associated with the degeneration of human intervertebral discs (IVDs). However, the pain-inducing mechanism in degenerating discs remains to be elucidated. Here, we identified a subtype of locally residing human nucleus pulposus cells (NPCs), generated by certain conditions in degenerating discs, that was associated with the onset of discogenic back pain. Single-cell transcriptomic analysis of human tissues showed a strong correlation between a specific cell subtype and the pain condition associated with the human degenerated disc, suggesting that they are pain-triggering. The application of IVD degeneration-associated exogenous stimuli to healthy NPCs in vitro recreated a pain-associated phenotype. These stimulated NPCs activated functional human iPSC-derived sensory neuron responses in an in vitro organ-chip model. Injection of stimulated NPCs into the healthy rat IVD induced local inflammatory responses and increased cold sensitivity and mechanical hypersensitivity. Our findings reveal a previously uncharacterized pain-inducing mechanism mediated by NPCs in degenerating IVDs. These findings could aid in the development of NPC-targeted therapeutic strategies for the clinically unmet need to attenuate discogenic LBP.

Targeted gene-and-host progenitor cell therapy for nonunion bone fracture repair.

Nonunion fractures present a challenge to orthopedics with no optimal solution. In-vivo DNA electroporation is a gene-delivery technique that can potentially accelerate regenerative processes. We hypothesized that in vivo electroporation of an osteogenic gene in a nonunion radius bone defect site would induce fracture repair. Nonunion fracture was created in the radii of C3H/HeN mice, into which a collagen sponge was placed. To allow for recruitment of host progenitor cells (HPCs) into the implanted sponge, the mice were housed for 10 days before electroporation. Mice were electroporated with either bone morphogenetic protein 9 (BMP-9) plasmid, Luciferase plasmid or injected with BMP-9 plasmid but not electroporated. In vivo bioluminescent imaging indicated that gene expression was localized to the defect site. Microcomputed tomography (µCT) and histological analysis of murine radii electroporated with BMP-9 demonstrated bone formation bridging the bone gap, whereas in the control groups the defect remained unbridged. Population of the implanted collagen sponge by HPCs transfected with the injected plasmid following electroporation was noted. Our data indicate that regeneration of nonunion bone defect can be attained by performing in vivo electroporation with an osteogenic gene combined with recruitment of HPCs. This gene therapy approach may pave the way for regeneration of other skeletal tissues.

iPSC-neural crest derived cells embedded in 3D printable bio-ink promote cranial bone defect repair.

Cranial bone loss presents a major clinical challenge and new regenerative approaches to address craniofacial reconstruction are in great demand. Induced pluripotent stem cell (iPSC) differentiation is a powerful tool to generate mesenchymal stromal cells (MSCs). Prior research demonstrated the potential of bone marrow-derived MSCs (BM-MSCs) and iPSC-derived mesenchymal progenitor cells via the neural crest (NCC-MPCs) or mesodermal lineages (iMSCs) to be promising cell source for bone regeneration. Overexpression of human recombinant bone morphogenetic protein (BMP)6 efficiently stimulates bone formation. The study aimed to evaluate the potential of iPSC-derived cells via neural crest or mesoderm overexpressing BMP6 and embedded in 3D printable bio-ink to generate viable bone graft alternatives for cranial reconstruction. Cell viability, osteogenic potential of cells, and bio-ink (Ink-Bone or GelXa) combinations were investigated in vitro using bioluminescent imaging. The osteogenic potential of bio-ink-cell constructs were evaluated in osteogenic media or nucleofected with BMP6 using qRT-PCR and in vitro µCT. For in vivo testing, two 2 mm circular defects were created in the frontal and parietal bones of NOD/SCID mice and treated with Ink-Bone, Ink-Bone + BM-MSC-BMP6, Ink-Bone + iMSC-BMP6, Ink-Bone + iNCC-MPC-BMP6, or left untreated. For follow-up, µCT was performed at weeks 0, 4, and 8 weeks. At the time of sacrifice (week 8), histological and immunofluorescent analyses were performed. Both bio-inks supported cell survival and promoted osteogenic differentiation of iNCC-MPCs and BM-MSCs in vitro. At 4 weeks, cell viability of both BM-MSCs and iNCC-MPCs were increased in Ink-Bone compared to GelXA. The combination of Ink-Bone with iNCC-MPC-BMP6 resulted in an increased bone volume in the frontal bone compared to the other groups at 4 weeks post-surgery. At 8 weeks, both iNCC-MPC-BMP6 and iMSC-MSC-BMP6 resulted in an increased bone volume and partial bone bridging between the implant and host bone compared to the other groups. The results of this study show the potential of NCC-MPC-incorporated bio-ink to regenerate frontal cranial defects. Therefore, this bio-ink-cell combination should be further investigated for its therapeutic potential in large animal models with larger cranial defects, allowing for 3D printing of the cell-incorporated material.

Gene-modified adult stem cells regenerate vertebral bone defect in a rat model.

Vertebral compression fractures (VCFs), the most common fragility fractures, account for approximately 700,000 injuries per year. Since open surgery involves morbidity and implant failure in the osteoporotic patient population, a new minimally invasive biological solution to vertebral bone repair is needed. Previously, we showed that adipose-derived stem cells (ASCs) overexpressing a BMP gene are capable of inducing spinal fusion in vivo. We hypothesized that a direct injection of ASCs, designed to transiently overexpress rhBMP6, into a vertebral bone void defect would accelerate bone regeneration. Porcine ASCs were isolated and labeled with lentiviral vectors that encode for the reporter gene luciferase (Luc) under constitutive (ubiquitin) or inductive (osteocalcin) promoters. The ASCs were first labeled with reporter genes and then nucleofected with an rhBMP6-encoding plasmid. Twenty-four hours later, bone void defects were created in the coccygeal vertebrae of nude rats. The ASC-BMP6 cells were suspended in fibrin gel (FG) and injected into the bone void. A control group was injected with FG alone. The regenerative process was monitored in vivo using microCT, and cell survival and differentiation were monitored using tissue specific reporter genes and bioluminescence imaging (BLI). The surgically treated vertebrae were harvested after 12 weeks and subjected to histological and immunohistochemical (against porcine vimentin) analyses. In vivo BLI detected Luc-expressing cells at the implantation site over a 12-week period. Beginning 2 weeks postoperatively, considerable defect repair was observed in the group treated with ASC-BMP6 cells. The rate of bone formation in the stem cell-treated group was two times faster than that in the FG-treated group, and bone volume at the end point was 2-fold compared to the control group. Twelve weeks after cell injection the bone volume within the void reached the volume measured in native vertebrae. Immunostaining against porcine vimentin indicated that the ASC-BMP6 cells contributed to new bone formation. Here we show the potential of injections of BMP-modified ASCs to repair vertebral bone defects in a rat model. Our results could pave the way to a novel approach for the biological treatment of traumatic and osteoporosis-related vertebral bone injuries.

Neural crest-derived mesenchymal progenitor cells enhance cranial allograft integration.

Replacement of lost cranial bone (partly mesodermal and partly neural crest-derived) is challenging and includes the use of nonviable allografts. To revitalize allografts, bone marrow-derived mesenchymal stromal cells (mesoderm-derived BM-MSCs) have been used with limited success. We hypothesize that coating of allografts with induced neural crest cell-mesenchymal progenitor cells (iNCC-MPCs) improves implant-to-bone integration in mouse cranial defects. Human induced pluripotent stem cells were reprogramed from dermal fibroblasts, differentiated to iNCCs and then to iNCC-MPCs. BM-MSCs were used as reference. Cells were labeled with luciferase (Luc2) and characterized for MSC consensus markers expression, differentiation, and risk of cellular transformation. A calvarial defect was created in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice and allografts were implanted, with or without cell coating. Bioluminescence imaging (BLI), microcomputed tomography (µCT), histology, immunofluorescence, and biomechanical tests were performed. Characterization of iNCC-MPC-Luc2 vs BM-MSC-Luc2 showed no difference in MSC markers expression and differentiation in vitro. In vivo, BLI indicated survival of both cell types for at least 8 weeks. At week 8, µCT analysis showed enhanced structural parameters in the iNCC-MPC-Luc2 group and increased bone volume in the BM-MSC-Luc2 group compared to controls. Histology demonstrated improved integration of iNCC-MPC-Luc2 allografts compared to BM-MSC-Luc2 group and controls. Human osteocalcin and collagen type 1 were detected at the allograft-host interphase in cell-seeded groups. The iNCC-MPC-Luc2 group also demonstrated improved biomechanical properties compared to BM-MSC-Luc2 implants and cell-free controls. Our results show an improved integration of iNCC-MPC-Luc2-coated allografts compared to BM-MSC-Luc2 and controls, suggesting the use of iNCC-MPCs as potential cell source for cranial bone repair.

A Translational Porcine Model for Human Cell-Based Therapies in the Treatment of Posttraumatic Osteoarthritis After Anterior Cruciate Ligament Injury.

BACKGROUND: There is a high incidence of posttraumatic osteoarthritis (PTOA) after anterior cruciate ligament (ACL) injury, and these injuries represent an enormous health care economic burden. In an effort to address this unmet clinical need, there has been increasing interest in cell-based therapies. PURPOSE: To establish a translational large animal model of PTOA and demonstrate the feasibility of intra-articular human cell-based interventions. STUDY DESIGN: Descriptive laboratory study. METHODS: Nine Yucatan mini-pigs underwent unilateral ACL transection and were monitored for up to 12 weeks after injury. Interleukin 1 beta (IL-1ß) levels and collagen breakdown were evaluated longitudinally using enzyme-linked immunosorbent assays of synovial fluid, serum, and urine. Animals were euthanized at 4 weeks (n = 3) or 12 weeks (n = 3) after injury, and injured and uninjured limbs underwent magnetic resonance imaging (MRI) and histologic analysis. At 2 days after ACL injury, an additional 3 animals received an intra-articular injection of 107 human bone marrow-derived mesenchymal stem cells (hBM-MSCs) combined with a fibrin carrier. These cells were labeled with the luciferase reporter gene (hBM-MSCs-Luc) as well as fluorescent markers and intracellular iron nanoparticles. These animals were euthanized on day 0 (n = 1) or day 14 (n = 2) after injection. hBM-MSC-Luc viability and localization were assessed using ex vivo bioluminescence imaging, fluorescence imaging, and MRI. RESULTS: PTOA was detected as early as 4 weeks after injury. At 12 weeks after injury, osteoarthritis could be detected grossly as well as on histologic analysis. Synovial fluid analysis showed elevation of IL-1ß shortly after ACL injury, with subsequent resolution by 2 weeks after injury. Collagen type II protein fragments were elevated in the synovial fluid and serum after injury. hBM-MSCs-Luc were detected immediately after injection and at 2 weeks after injection using fluorescence imaging, MRI, and bioluminescence imaging. CONCLUSION: This study demonstrates the feasibility of reproducing the chondral changes, intra-articular cytokine alterations, and body fluid biomarker findings consistent with PTOA after ACL injury in a large animal model. Furthermore, we have demonstrated the ability of hBM-MSCs to survive and express transgene within the knee joint of porcine hosts without immunosuppression for at least 2 weeks. CLINICAL RELEVANCE: This model holds great potential to significantly contribute to investigations focused on the development of cell-based therapies for human ACL injury-associated PTOA in the future (see Appendix Figure A1, available online).

Teriparatide (recombinant parathyroid hormone 1-34) enhances bone allograft integration in a clinically relevant pig model of segmental mandibulectomy.

Massive craniofacial bone loss poses a clinical challenge to maxillofacial surgeons. Structural bone allografts are readily available at tissue banks but are rarely used due to a high failure rate. Previous studies showed that intermittent administration of recombinant parathyroid hormone (rPTH) enhanced integration of allografts in a murine model of calvarial bone defect. To evaluate its translational potential, the hypothesis that rPTH would enhance healing of a mandibular allograft in a clinically relevant large animal model of mandibulectomy was tested. Porcine bone allografts were implanted into a 5-cm-long continuous mandible bone defect in six adult Yucatan minipigs, which were randomized to daily intramuscular injections of rPTH (1.75 µg/kg) and placebo (n = 3). Blood tests were performed on Day 56 preoperation, Day 0 and on Day 56 postoperation. Eight weeks after the surgery, bone healing was analyzed using high-resolution X-ray imaging (Faxitron and micro computed tomography [CT]) and three-point bending biomechanical testing. The results showed a significant 2.6-fold rPTH-induced increase in bone formation (p = 0.02). Biomechanically, the yield failure properties of the healed mandibles were significantly higher in the rPTH group (yield load: p < 0.05; energy to yield: p < 0.01), and the post-yield displacement and energy were higher in the placebo group (p < 0.05), suggesting increased mineralized integration of the allograft in the rPTH group. In contrast to similar rPTH therapy studies in dogs, no signs of hypercalcemia, hyperphosphatemia, or inflammation were detected. Taken together, we provide initial evidence that rPTH treatment enhances mandibular allograft healing in a clinically relevant large animal model.

Optimization of a rat lumbar IVD degeneration model for low back pain.

INTRODUCTION: Intervertebral disc (IVD) degeneration is often associated with low back pain and radiating leg pain. The purpose of this study is to develop a reproducible and standardized preclinical model of painful lumbar IVD degeneration by evaluation of structural and behavioral changes in response to IVD injury with increasing needle sizes. This model can be used to develop new therapies for IVD degeneration. METHODS: Forty-five female Sprague Dawley rats underwent anterior lumbar disc needle puncture at levels L4-5 and L5-6 under fluoroscopic guidance. Animals were randomly assigned to four different experimental groups: needle sizes of 18 Gauge (G), 21G, 23G, and sham control. To monitor the progression of IVD degeneration and pain, the following methods were employed: µMRI, qRT-PCR, histology, and biobehavioral analysis. RESULTS: T1- and T2-weighted µMRI analysis showed a correlation between the degree of IVD degeneration and needle diameter, with the most severe degeneration in the 18G group. mRNA expression of markers for IVD degeneration markers were dysregulated in the 18G and 21G groups, while pro-nociceptive markers were increased in the 18G group only. Hematoxylin and Eosin (H&E) and Alcian Blue/Picrosirius Red staining confirmed the most pronounced IVD degeneration in the 18G group. Randall-Selitto and von Frey tests showed increased hindpaw sensitivity in the 18G group. CONCLUSION: Our findings demonstrate that anterior disc injury with an 18G needle creates severe IVD degeneration and mechanical hypersensitivity, while the 21G needle results in moderate degeneration with no increased pain sensitivity. Therefore, needle sizes should be selected depending on the desired phenotype for the pre-clinical model.

Vibrational testing of trabecular bone architectures using rapid prototype models.

The purpose of this study was to investigate if standard analysis of the vibrational characteristics of trabecular architectures can be used to detect changes in the mechanical properties due to progressive bone loss. A cored trabecular specimen from a human lumbar vertebra was microCT scanned and a three-dimensional, virtual model in stereolithography (STL) format was generated. Uniform bone loss was simulated using a surface erosion algorithm. Rapid prototype (RP) replicas were manufactured from these virtualised models with 0%, 16% and 42% bone loss. Vibrational behaviour of the RP replicas was evaluated by performing a dynamic compression test through a frequency range using an electro-dynamic shaker. The acceleration and dynamic force responses were recorded and fast Fourier transform (FFT) analyses were performed to determine the response spectrum. Standard resonant frequency analysis and damping factor calculations were performed. The RP replicas were subsequently tested in compression beyond failure to determine their strength and modulus. It was found that the reductions in resonant frequency with increasing bone loss corresponded well with reductions in apparent stiffness and strength. This suggests that structural dynamics has the potential to be an alternative diagnostic technique for osteoporosis, although significant challenges must be overcome to determine the effect of the skin/soft tissue interface, the cortex and variabilities associated with in vivo testing.

In Vivo Imaging of Exogenous Progenitor Cells in Tendon Regeneration via Superparamagnetic Iron Oxide Particles.

BACKGROUND: Although tendon injuries and repairs are common, treatment of these injuries has limitations. The application of mesenchymal progenitor cells (MPCs) is increasingly used to optimize the biological process of tendon repair healing. However, clinically relevant technologies that effectively assess the localization of exogenous MPCs in vivo are lacking. HYPOTHESIS: Exogenous MPCs labeled with superparamagnetic iron oxide (SPIO) particles would allow monitoring of the localization and retention of cells within the site of implantation via magnetic resonance imaging (MRI) without negatively affecting cell survival or differentiation. STUDY DESIGN: Descriptive laboratory study. METHODS: Genetically modified C3H10T1/2 MPCs engineered to express luciferase (Luc+) reporter gene were implanted into surgically created Achilles tendon defects of 10 athymic nude rats (Hsd:RH-Foxn1rnu). Of these animals, 5 animals received Luc+ C3H10T1/2 MPCs colabeled with SPIO nanoparticles (+SPIO). These 2 groups of animals then underwent optical imaging with quantification of bioluminescence and MRI at 7, 14, and 28 days after surgery. Statistical analysis was conducted by use of 2-way analysis of variance. At 28 days after surgery, animals were euthanized and the treated limbs underwent histologic analysis. RESULTS: Optical imaging demonstrated that the implanted cells not only survived but also proliferated in vivo, and these cells remained viable for at least 4 weeks after implantation. In addition, SPIO labeling did not appear to affect MPC survival or proliferation, as assessed by quantitative bioluminescence imaging (P > .05, n = 5). MRI demonstrated that SPIO labeling was an effective method to monitor cell localization, retention, and viability for at least 4 weeks after implantation. Histologic and immunofluorescence analyses of the repaired tendon defect sites demonstrated tenocyte-like labeled cells, suggesting that cell differentiation was not affected by labeling the cells with the SPIO nanoparticles. CONCLUSION: MRI of exogenous MPCs labeled with SPIO particles allows for effective in vivo assessments of cell localization and retention in the setting of tendon regeneration for at least 4 weeks after implantation. This SPIO labeling does not appear to impair cell survival, transgene expression, or differentiation. CLINICAL RELEVANCE: SPIO labeling of MPCs appears to be safe for in vivo assessments of MPCs in tendon regeneration therapies and may be used for future clinical investigations of musculoskeletal regenerative medicine.