A theranostic 3D ultrasound imaging system for high resolution image-guided therapy.

Microbubble contrast agents are a diagnostic tool with broad clinical impact and an increasing number of indications. Many therapeutic applications have also been identified. Yet, technologies for ultrasound guidance of microbubble-mediated therapy are limited. In particular, arrays that are capable of implementing and imaging microbubble-based therapy in three dimensions in real-time are lacking. We propose a system to perform and monitor microbubble-based therapy, capable of volumetric imaging over a large field-of-view. To propel the promise of the theranostic treatment strategies forward, we have designed and tested a unique array and system for 3D ultrasound guidance of microbubble-based therapeutic protocols based on the frequency, temporal and spatial requirements. Methods: Four 256-channel plane wave scanners (Verasonics, Inc, WA, USA) were combined to control a 1024-element planar array with 1.3 and 2.5 MHz therapeutic and imaging transmissions, respectively. A transducer aperture of ~40×15 mm was selected and Field II was applied to evaluate the point spread function. In vitro experiments were performed on commercial and custom phantoms to assess the spatial resolution, image contrast and microbubble-enhanced imaging capabilities. Results: We found that a 2D array configuration with 64 elements separated by λ-pitch in azimuth and 16 elements separated by 1.5λ-pitch in elevation ensured the required flexibility. This design, of 41.6 mm × 16 mm, thus provided both an extended field-of-view, up to 11 cm x 6 cm at 10 cm depth and steering of ±18° in azimuth and ±12° in elevation. At a depth of 16 cm, we achieved a volume imaging rate of 60 Hz, with a contrast ratio and resolution, respectively, of 19 dB, 0.8 mm at 3 cm and 20 dB and 2.1 mm at 12.5 cm. Conclusion: A single 2D array for both imaging and therapeutics, integrated with a 1024 channel scanner can guide microbubble-based therapy in volumetric regions of interest.

Encapsulation of Human-Bone-Marrow-Derived Mesenchymal Stem Cells in Small Alginate Beads Using One-Step Emulsification by Internal Gelation: In Vitro, and In Vivo Evaluation in Degenerate Intervertebral Disc Model.

Cell microencapsulation in gel beads contributes to many biomedical processes and pharmaceutical applications. Small beads (<300 µm) offer distinct advantages, mainly due to improved mass transfer and mechanical strength. Here, we describe, for the first time, the encapsulation of human-bone-marrow-derived mesenchymal stem cells (hBM-MSCs) in small-sized microspheres, using one-step emulsification by internal gelation. Small (127−257 µm) high-mannuronic-alginate microspheres were prepared at high agitation rates (800−1000 rpm), enabling control over the bead size and shape. The average viability of encapsulated hBM-MSCs after 2 weeks was 81 ± 4.3% for the higher agitation rates. hBM-MSC-loaded microspheres seeded within a glycosaminoglycan (GAG) analogue, which was previously proposed as a mechanically equivalent implant for degenerate discs, kept their viability, sphericity, and integrity for at least 6 weeks. A preliminary in vivo study of hBM-MSC-loaded microspheres implanted (via a GAG-analogue hydrogel) in a rat injured intervertebral disc model demonstrated long-lasting viability and biocompatibility for at least 8 weeks post-implantation. The proposed method offers an effective and reproducible way to maintain long-lasting viability in vitro and in vivo. This approach not only utilizes the benefits of a simple, mild, and scalable method, but also allows for the easy control of the bead size and shape by the agitation rate, which, overall, makes it a very attractive platform for regenerative-medicine applications.

Applications of Ultrasound-Mediated Gene Delivery in Regenerative Medicine.

Research on the capability of non-viral gene delivery systems to induce tissue regeneration is a continued effort as the current use of viral vectors can present with significant limitations. Despite initially showing lower gene transfection and gene expression efficiencies, non-viral delivery methods continue to be optimized to match that of their viral counterparts. Ultrasound-mediated gene transfer, referred to as sonoporation, occurs by the induction of transient membrane permeabilization and has been found to significantly increase the uptake and expression of DNA in cells across many organ systems. In addition, it offers a more favorable safety profile compared to other non-viral delivery methods. Studies have shown that microbubble-enhanced sonoporation can elicit significant tissue regeneration in both ectopic and disease models, including bone and vascular tissue regeneration. Despite this, no clinical trials on the use of sonoporation for tissue regeneration have been conducted, although current clinical trials using sonoporation for other indications suggest that the method is safe for use in the clinical setting. In this review, we describe the pre-clinical studies conducted thus far on the use of sonoporation for tissue regeneration. Further, the various techniques used to increase the effectiveness and duration of sonoporation-induced gene transfer, as well as the obstacles that may be currently hindering clinical translation, are explored.

PTH-Induced Bone Regeneration and Vascular Modulation Are Both Dependent on Endothelial Signaling.

The use of a bone allograft presents a promising approach for healing nonunion fractures. We have previously reported that parathyroid hormone (PTH) therapy induced allograft integration while modulating angiogenesis at the allograft proximity. Here, we hypothesize that PTH-induced vascular modulation and the osteogenic effect of PTH are both dependent on endothelial PTH receptor-1 (PTHR1) signaling. To evaluate our hypothesis, we used multiple transgenic mouse lines, and their wild-type counterparts as a control. In addition to endothelial-specific PTHR1 knock-out mice, we used mice in which PTHR1 was engineered to be constitutively active in collagen-1α+ osteoblasts, to assess the effect of PTH signaling activation exclusively in osteoprogenitors. To characterize resident cell recruitment and osteogenic activity, mice in which the Luciferase reporter gene is expressed under the Osteocalcin promoter (Oc-Luc) were used. Mice were implanted with calvarial allografts and treated with either PTH or PBS. A micro-computed tomography-based structural analysis indicated that the induction of bone formation by PTH, as observed in wild-type animals, was not maintained when PTHR1 was removed from endothelial cells. Furthermore, the induction of PTH signaling exclusively in osteoblasts resulted in significantly less bone formation compared to systemic PTH treatment, and significantly less osteogenic activity was measured by bioluminescence imaging of the Oc-Luc mice. Deletion of the endothelial PTHR1 significantly decreased the PTH-induced formation of narrow blood vessels, formerly demonstrated in wild-type mice. However, the exclusive activation of PTH signaling in osteoblasts was sufficient to re-establish the observed PTH effect. Collectively, our results show that endothelial PTHR1 signaling plays a key role in PTH-induced osteogenesis and has implications in angiogenesis.

Intradiscal quantitative chemical exchange saturation transfer MRI signal correlates with discogenic pain in human patients.

Low back pain (LBP) is often a result of a degenerative process in the intervertebral disc. The precise origin of discogenic pain is diagnosed by the invasive procedure of provocative discography (PD). Previously, we developed quantitative chemical exchange saturation transfer (qCEST) magnetic resonance imaging (MRI) to detect pH as a biomarker for discogenic pain. Based on these findings we initiated a clinical study with the goal to evaluate the correlation between qCEST values and PD results in LBP patients. Twenty five volunteers with chronic low back pain were subjected to T2-weighted (T2w) and qCEST MRI scans followed by PD. A total of 72 discs were analyzed. The average qCEST signal value of painful discs was significantly higher than non-painful discs (p = 0.012). The ratio between qCEST and normalized T2w was found to be significantly higher in painful discs compared to non-painful discs (p = 0.0022). A receiver operating characteristics (ROC) analysis indicated that qCEST/T2w ratio could be used to differentiate between painful and non-painful discs with 78% sensitivity and 81% specificity. The results of the study suggest that qCEST could be used for the diagnosis of discogenic pain, in conjunction with the commonly used T2w scan.

Stem Cells and Exosomes: New Therapies for Intervertebral Disc Degeneration.

Intervertebral disc degeneration (IVDD) occurs as a result of an imbalance of the anabolic and catabolic processes in the intervertebral disc, leading to an alteration in the composition of the extracellular matrix (ECM), loss of nucleus pulposus (NP) cells, excessive oxidative stress and inflammation. Degeneration of the IVD occurs naturally with age, but mechanical trauma, lifestyle factors and certain genetic abnormalities can increase the likelihood of symptomatic disease progression. IVDD, often referred to as degenerative disc disease (DDD), poses an increasingly substantial financial burden due to the aging population and increasing incidence of obesity in the United States. Current treatments for IVDD include pharmacological and surgical interventions, but these lack the ability to stop the progression of disease and restore the functionality of the IVD. Biological therapies have been evaluated but show varying degrees of efficacy in reversing disc degeneration long-term. Stem cell-based therapies have shown promising results in the regeneration of the IVD, but face both biological and ethical limitations. Exosomes play an important role in intercellular communication, and stem cell-derived exosomes have been shown to maintain the therapeutic benefit of their origin cells without the associated risks. This review highlights the current state of research on the use of stem-cell derived exosomes in the treatment of IVDD.

A comprehensive tool box for large animal studies of intervertebral disc degeneration.

Preclinical studies involving large animal models aim to recapitulate the clinical situation as much as possible and bridge the gap from benchtop to bedside. To date, studies investigating intervertebral disc (IVD) degeneration and regeneration in large animal models have utilized a wide spectrum of methodologies for outcome evaluation. This paper aims to consolidate available knowledge, expertise, and experience in large animal preclinical models of IVD degeneration to create a comprehensive tool box of anatomical and functional outcomes. Herein, we present a Large Animal IVD Scoring Algorithm based on three scales: macroscopic (gross morphology, imaging, and biomechanics), microscopic (histological, biochemical, and biomolecular analyses), and clinical (neurologic state, mobility, and pain). The proposed algorithm encompasses a stepwise evaluation on all three scales, including spinal pain assessment, and relevant structural and functional components of IVD health and disease. This comprehensive tool box was designed for four commonly used preclinical large animal models (dog, pig, goat, and sheep) in order to facilitate standardization and applicability. Furthermore, it is intended to facilitate comparison across studies while discerning relevant differences between species within the context of outcomes with the goal to enhance veterinary clinical relevance as well. Current major challenges in pre-clinical large animal models for IVD regeneration are highlighted and insights into future directions that may improve the understanding of the underlying pathologies are discussed. As such, the IVD research community can deepen its exploration of the molecular, cellular, structural, and biomechanical changes that occur with IVD degeneration and regeneration, paving the path for clinically relevant therapeutic strategies.

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.

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.

Human iPSCs can be differentiated into notochordal cells that reduce intervertebral disc degeneration in a porcine model.

Introduction: As many as 80% of the adult population experience back pain at some point in their lifetimes. Previous studies have indicated a link between back pain and intervertebral disc (IVD) degeneration. Despite decades of research, there is an urgent need for robust stem cell therapy targeting underlying causes rather than symptoms. It has been proposed that notochordal cells (NCs) appear to be the ideal cell type to regenerate the IVD: these cells disappear in humans as they mature, are replaced by nucleus pulposus (NP) cells, and their disappearance correlates with the initiation of degeneration of the disc. Human NCs are in short supply, thus here aimed for generation of notochordal-like cells from induced pluripotent cells (iPSCs). Methods: Human iPSCs were generated from normal dermal fibroblasts by transfecting plasmids encoding for six factors: OCT4, SOX2, KLF4, L-MYC, LIN28, and p53 shRNA. Then the iPSCs were treated with GSK3i to induce differentiation towards Primitive Streak Mesoderm (PSM). The differentiation was confirmed by qRT-PCR and immunofluorescence. PSM cells were transfected with Brachyury (Br)-encoding plasmid and the cells were encapsulated in Tetronic-tetraacrylate-fibrinogen (TF) hydrogel that mimics the NP environment (G'=1kPa), cultured in hypoxic conditions (2% O2) and with specifically defined growth media. The cells were also tested in vivo in a large animal model. IVD degeneration was induced after an annular puncture in pigs, 4 weeks later the cells were injected and IVDs were analyzed at 12 weeks after the injury using MRI, gene expression analysis and histology. Results: After short-term exposure of iPSCs to GSK3i there was a significant change in cell morphology, Primitive Streak Mesoderm (PSM) markers (Brachyury, MIXL1, FOXF1) were upregulated and markers of pluripotency (Nanog, Oct4, Sox2) were downregulated, both compared to the control group. PSM cells nucleofected with Br (PSM-Br) cultured in TF hydrogels retained the NC phenotype consistently for up to 8 weeks, as seen in the gene expression analysis. PSM-Br cells were co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs) which, with time, expressed the NC markers in higher levels, however the levels of expression in BM-MSCs alone did not change. Higher expression of NC and NP marker genes in human BM-MSCs was found to be induced by iNC-condition media (iNC-CM) than porcine NC-CM. The annular puncture induced IVD degeneration as early as 2 weeks after the procedure. The injected iNCs were detected in the degenerated discs after 8 weeks in vivo. The iNC-treated discs were found protected from degeneration. This was evident in histological analysis and changes in the pH levels, indicative of degeneration state of the discs, observed using qCEST MRI. Immunofluorescence stains show that their phenotype was consistent with the in vitro study, namely they still expressed the notochordal markers Keratin 18, Keratin 19, Noto and Brachyury. Conclusion: In the present study, we report a stepwise differentiation method to generate notochordal cells from human iPSCs. These cells not only demonstrate a sustainable notochordal cell phenotype in vitro and in vivo, but also show the functionality of notochordal cells and have protective effect in case of induced disc degeneration and prevent the change in the pH level of the injected IVDs. The mechanism of this effect could be suggested via the paracrine effect on resident cells, as it was shown in the in vitro studies with MSCs.

Bone-chip system to monitor osteogenic differentiation using optical imaging.

Human organoids and organ-on-chip systems to predict human responses to new therapies and for the understanding of disease mechanisms are being more commonly used in translational research. We have developed a bone-chip system to study osteogenic differentiation in vitro, coupled with optical imaging approach which provides the opportunity of monitoring cell survival, proliferation and differentiation in vitro without the need to terminate the culture. We used the mesenchymal stem cell (MSC) line over-expressing bone morphogenetic protein-2 (BMP-2), under Tet-Off system, and luciferase reporter gene under constitutive promoter. Cells were seeded on chips and supplemented with osteogenic medium. Flow of media was started 24 h later, while static cultures were performed using media reservoirs. Cells grown on the bone-chips under constant flow of media showed enhanced survival/proliferation, comparing to the cells grown in static conditions; luciferase reporter gene expression and activity, reflecting the cell survival and proliferation, was quantified using bioluminescence imaging and a significant advantage to the flow system was observed. In addition, the flow had positive effect on osteogenic differentiation, when compared with static cultures. Quantitative fluorescent imaging, performed using the osteogenic extra-cellular matrix-targeted probes, showed higher osteogenic differentiation of the cells under the flow conditions. Gene expression analysis of osteogenic markers confirmed the osteogenic differentiation of the MSC-BMP2 cells. Immunofluorescent staining performed against the Osteocalcin, Col1, and BSP markers illustrated robust osteogenic differentiation in the flow culture and lessened differentiation in the static culture. To sum, the bone-chip allows monitoring cell survival, proliferation, and osteogenic differentiation using optical imaging.

Molecular pain markers correlate with pH-sensitive MRI signal in a pig model of disc degeneration.

Intervertebral disc (IVD) degeneration is a leading cause of chronic low back pain that affects millions of people every year. Yet identification of the specific IVD causing this pain is based on qualitative visual interpretation rather than objective findings. One possible approach to diagnosing pain-associated IVD could be to identify acidic IVDs, as decreased pH within an IVD has been postulated to mediate discogenic pain. We hypothesized that quantitative chemical exchange saturation transfer (qCEST) MRI could detect pH changes in IVDs, and thence be used to diagnose pathologically painful IVDs objectively and noninvasively. To test this hypothesis, a surgical model of IVD degeneration in Yucatan minipigs was used. Direct measurement of pH inside the degenerated IVDs revealed a significant drop in pH after degeneration, which correlated with a significant increase in the qCEST signal. Gene analysis of harvested degenerated IVDs revealed significant upregulation of pain-, nerve- and inflammatory-related markers after IVD degeneration. A strong positive correlation was observed between the expression of pain markers and the increase in the qCEST signal. Collectively, these findings suggest that this approach might be used to identify which IVD is causing low back pain, thereby providing valuable guidance for pain and surgical management.

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.

Latest advances in intervertebral disc development and progenitor cells.

This paper is a concise review aiming to assemble the most relevant topics presented by the authors at ORS-Philadelphia Spine Research Society Fourth International Spine Research Symposium. It centers on the latest advances in disc development, its main structural entities, and the populating cells, with emphasis on the advances in pivotal molecular pathways responsible for forming the intervertebral discs (IVD). The objective of finding and emphasizing pathways and mechanisms that function to control tissue formation is to identify and to explore modifications occurring during normal aging, disease, and tissue repair. Thus, to comprehend that the cellular and molecular basis of tissue degeneration are crucial in the study of the dynamic interplay that includes cell-cell communication, gene regulation, and growth factors required to form a healthy and functional tissue during normal development.

Semiautomated Longitudinal Microcomputed Tomography-based Quantitative Structural Analysis of a Nude Rat Osteoporosis-related Vertebral Fracture Model.

Osteoporosis-related vertebral compression fractures (OVCFs) are a common and clinically unmet need with increasing prevalence as the world population ages. Animal OVCF models are essential to the preclinical development of translational tissue engineering strategies. While a number of models currently exist, this protocol describes an optimized method for inducing multiple highly reproducible vertebral defects in a single nude rat. A novel longitudinal semiautomated microcomputed tomography (µCT)-based quantitative structural analysis of the vertebral defects is also detailed. Briefly, rats were imaged at multiple time points post-op. The day 1 scan was reoriented to a standard position, and a standard volume of interest was defined. Subsequent µCT scans of each rat were automatically registered to the day 1 scan so the same volume of interest was then analyzed to assess for new bone formation. This versatile approach can be adapted to a variety of other models where longitudinal imaging-based analysis could benefit from precise 3D semiautomated alignment. Taken together, this protocol describes a readily quantifiable and easily reproducible system for osteoporosis and bone research. The suggested protocol takes 4 months to induce osteoporosis in nude ovariectomized rats and between 2.7 and 4 h to generate, image, and analyze two vertebral defects, depending on tissue size and equipment.

In situ bone tissue engineering via ultrasound-mediated gene delivery to endogenous progenitor cells in mini-pigs.

More than 2 million bone-grafting procedures are performed each year using autografts or allografts. However, both options carry disadvantages, and there remains a clear medical need for the development of new therapies for massive bone loss and fracture nonunions. We hypothesized that localized ultrasound-mediated, microbubble-enhanced therapeutic gene delivery to endogenous stem cells would induce efficient bone regeneration and fracture repair. To test this hypothesis, we surgically created a critical-sized bone fracture in the tibiae of Yucatán mini-pigs, a clinically relevant large animal model. A collagen scaffold was implanted in the fracture to facilitate recruitment of endogenous mesenchymal stem/progenitor cells (MSCs) into the fracture site. Two weeks later, transcutaneous ultrasound-mediated reporter gene delivery successfully transfected 40% of cells at the fracture site, and flow cytometry showed that 80% of the transfected cells expressed MSC markers. Human bone morphogenetic protein-6 (BMP-6) plasmid DNA was delivered using ultrasound in the same animal model, leading to transient expression and secretion of BMP-6 localized to the fracture area. Micro-computed tomography and biomechanical analyses showed that ultrasound-mediated BMP-6 gene delivery led to complete radiographic and functional fracture healing in all animals 6 weeks after treatment, whereas nonunion was evident in control animals. Collectively, these findings demonstrate that ultrasound-mediated gene delivery to endogenous mesenchymal progenitor cells can effectively treat nonhealing bone fractures in large animals, thereby addressing a major orthopedic unmet need and offering new possibilities for clinical translation.

Systemic administration of mesenchymal stem cells combined with parathyroid hormone therapy synergistically regenerates multiple rib fractures.

BACKGROUND: A devastating condition that leads to trauma-related morbidity, multiple rib fractures, remain a serious unmet clinical need. Systemic administration of mesenchymal stem cells (MSCs) has been shown to regenerate various tissues. We hypothesized that parathyroid hormone (PTH) therapy would enhance MSC homing and differentiation, ultimately leading to bone formation that would bridge rib fractures. METHODS: The combination of human MSCs (hMSCs) and a clinically relevant PTH dose was studied using immunosuppressed rats. Segmental defects were created in animals' fifth and sixth ribs. The rats were divided into four groups: a negative control group, in which animals received vehicle alone; the PTH-only group, in which animals received daily subcutaneous injections of 4 µg/kg teriparatide, a pharmaceutical derivative of PTH; the hMSC-only group, in which each animal received five injections of 2 × 106 hMSCs; and the hMSC + PTH group, in which animals received both treatments. Longitudinal in vivo monitoring of bone formation was performed biweekly using micro-computed tomography (µCT), followed by histological analysis. RESULTS: Fluorescently-dyed hMSCs were counted using confocal microscopy imaging of histological samples harvested 8 weeks after surgery. PTH significantly augmented the number of hMSCs that homed to the fracture site. Immunofluorescence of osteogenic markers, osteocalcin and bone sialoprotein, showed that PTH induced cell differentiation in both exogenously administered cells and resident cells. µCT scans revealed a significant increase in bone volume only in the hMSC + PTH group, beginning by the 4th week after surgery. Eight weeks after surgery, 35% of ribs in the hMSC + PTH group had complete bone bridging, whereas there was complete bridging in only 6.25% of ribs (one rib) in the PTH-only group and in none of the ribs in the other groups. Based on the µCT scans, biomechanical analysis using the micro-finite element method demonstrated that the healed ribs were stiffer than intact ribs in torsion, compression, and bending simulations, as expected when examining bone callus composed of woven bone. CONCLUSIONS: Administration of both hMSCs and PTH worked synergistically in rib fracture healing, suggesting this approach may pave the way to treat multiple rib fractures as well as additional fractures in various anatomical sites.

Teriparatide attenuates scarring around murine cranial bone allograft via modulation of angiogenesis.

Nearly all bone fractures in humans can deteriorate into a non-union fracture, often due to formation of fibrotic tissue. Cranial allogeneic bone grafts present a striking example: although seemingly attractive for craniofacial reconstructions, they often fail due to fibrosis at the host-graft junction, which physically prevents the desired bridging of bone between the host and graft and revitalization of the latter. In the present study we show that intermittent treatment with recombinant parathyroid hormone-analogue (teriparatide) modulates neovascularization feeding in the graft surroundings, consequently reducing fibrosis and scar tissue formation and facilitates osteogenesis. Longitudinal inspection of the vascular tree feeding the allograft has revealed that teriparatide induces formation of small-diameter vessels in the 1st week after surgery; by the 2nd week, abundant formation of small-diameter blood vessels was detected in untreated control animals, but far less in teriparatide-treated mice, although in total, more blood capillaries were detected in the animals that were given teriparatide. By that time point we observed expression of the profibrogenic mediator TGF-ß in untreated animals, but negligible expression in the teriparatide-treated mice. To evaluate the formation of scar tissue, we utilized a magnetization transfer contrast MRI protocol to differentiate osteoid tissue from scar tissue, based on the characterization of collagen fibers. Using this method we found that significantly more bone matrix was formed in animals given teriparatide than in control animals. Altogether, our findings show how teriparatide diminishes scarring, ultimately leading to superior bone graft integration.

Isolation and characterization of mesenchymal stromal progenitors from the temporomandibular joint disc.

Disorders of the temporomandibular joint (TMJ) complex affect 6-12% of the population; the joint's disc is usually involved. Tissue engineering and regenerative medicine may constitute a promising therapeutic approach, with resident stromal progenitor cells a key factor in the process. We hypothesized that the TMJ disc (TMJD) contains multipotent stromal progenitors that may play an important role in regeneration of the disc. TMJD cells were cultured and evaluated for growth kinetics and colony-forming units (CFUs). Single cell-derived clones were isolated and induced to differentiate toward the osteogenic, adipogenic and chondrogenic lineages by culturing in various induction media. Flow cytometry was used to identify multipotent stromal cell surface markers in additional cell samples, and reverse transcription-polymerase chain reaction (RT-PCR) was used to determine gene expression patterns within isolated cells. High numbers of CFUs were observed, indicating cell self-renewal. Biochemical assays showed significantly higher alkaline phosphatase (ALP) activity, lipid droplet concentration and glycosaminoglycan levels in cells cultured in osteogenic, adipogenic and chondrogenic induction medium, respectively. Approximately 1% of the total cell population demonstrated the capability to differentiate into all three mesenchymal lineages. Chondrogenic gene levels within TMJD-derived cells were significantly reduced in passaged culture. Our results support the hypothesis that multipotent stromal progenitor cells populate the TMJD and possess proliferation and differentiation capabilities. These cells may contribute to the regeneration potential of dysfunctional tissue and become the primary component in future attempts at tissue engineering or regeneration of this complex. Copyright © 2015 John Wiley & Sons, Ltd.

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.