Synergistic effect of umbilical cord extracellular vesicles and rhBMP-2 to enhance the regeneration of a metaphyseal femoral defect in osteoporotic rats.

BACKGROUND: The aim of this study was to evaluate potential synergistic effects of a single, local application of human umbilical cord MSC-derived sEVs in combination with a low dose of recombinant human rhBMP-2 to promote the regeneration of a metaphyseal femoral defect in an osteoporotic rat model. METHODS: 6 weeks after induction of osteoporosis by bilateral ventral ovariectomy and administration of a special diet, a total of 64 rats underwent a distal femoral metaphyseal osteotomy using a manual Gigli wire saw. Defects were stabilized with an adapted Y-shaped mini-locking plate and were subsequently treated with alginate only, or alginate loaded with hUC-MSC-sEVs (2 × 109), rhBMP-2 (1.5 µg), or a combination of sEVs and rhBMP-2 (n = 16 for each group). 6 weeks post-surgery, femora were evaluated by µCT, descriptive histology, and biomechanical testing. RESULTS: Native radiographs and µCT analysis confirmed superior bony union with callus formation after treatment with hUC-MSC-sEVs in combination with a low dose of rhBMP-2. This finding was further substantiated by histology, showing robust defect consolidation 6 weeks after treatment. Torsion testing of the explanted femora revealed increased stiffness after application of both, rhBMP-2 alone, or in combination with sEVs, whereas torque was only significantly increased after treatment with rhBMP-2 together with sEVs. CONCLUSION: The present study demonstrates that the co-application of hUC-MSC-sEVs can improve the efficacy of rhBMP-2 to promote the regeneration of osteoporotic bone defects.

Engineering of extracellular matrix from human iPSC-mesenchymal progenitors to enhance osteogenic capacity of human bone marrow stromal cells independent of their age.

Regeneration of bone defects is often limited due to compromised bone tissue physiology. Previous studies suggest that engineered extracellular matrices enhance the regenerative capacity of mesenchymal stromal cells. In this study, we used human-induced pluripotent stem cells, a scalable source of young mesenchymal progenitors (hiPSC-MPs), to generate extracellular matrix (iECM) and test its effects on the osteogenic capacity of human bone-marrow mesenchymal stromal cells (BMSCs). iECM was deposited as a layer on cell culture dishes and into three-dimensional (3D) silk-based spongy scaffolds. After decellularization, iECM maintained inherent structural proteins including collagens, fibronectin and laminin, and contained minimal residual DNA. Young adult and aged BMSCs cultured on the iECM layer in osteogenic medium exhibited a significant increase in proliferation, osteogenic marker expression, and mineralization as compared to tissue culture plastic. With BMSCs from aged donors, matrix mineralization was only detected when cultured on iECM, but not on tissue culture plastic. When cultured in 3D iECM/silk scaffolds, BMSCs exhibited significantly increased osteogenic gene expression levels and bone matrix deposition. iECM layer showed a similar enhancement of aged BMSC proliferation, osteogenic gene expression, and mineralization compared with extracellular matrix layers derived from young adult or aged BMSCs. However, iECM increased osteogenic differentiation and decreased adipocyte formation compared with single protein substrates including collagen and fibronectin. Together, our data suggest that the microenvironment comprised of iECM can enhance the osteogenic activity of BMSCs, providing a bioactive and scalable biomaterial strategy for enhancing bone regeneration in patients with delayed or failed bone healing.

Pulsed Electromagnetic Fields (PEMF)-Physiological Response and Its Potential in Trauma Treatment.

Environmental biophysical interactions are recognized to play an essential part in the human biological processes associated with trauma recovery. Many studies over several decades have furthered our understanding of the effects that Pulsed Electromagnetic Fields (PEMF) have on the human body, as well as on cellular and biophysical systems. These investigations have been driven by the observed positive clinical effects of this non-invasive treatment on patients, mainly in orthopedics. Unfortunately, the diversity of the various study setups, with regard to physical parameters, molecular and cellular response, and clinical outcomes, has made it difficult to interpret and evaluate commonalities, which could, in turn, lead to finding an underlying mechanistic understanding of this treatment modality. In this review, we give a birds-eye view of the vast landscape of studies that have been published on PEMF, presenting the reader with a scaffolded summary of relevant literature starting from categorical literature reviews down to individual studies for future research studies and clinical use. We also highlight discrepancies within the many diverse study setups to find common reporting parameters that can lead to a better universal understanding of PEMF effects.

Wavelength-Dependent Effects of Photobiomodulation for Wound Care in Diabetic Wounds.

Photobiomodulation, showing positive effects on wound healing processes, has been performed mainly with lasers in the red/infrared spectrum. Light of shorter wavelengths can significantly influence biological systems. This study aimed to evaluate and compare the therapeutic effects of pulsed LED light of different wavelengths on wound healing in a diabetic (db/db) mouse excision wound model. LED therapy by Repuls was applied at either 470 nm (blue), 540 nm (green) or 635 nm (red), at 40 mW/cm2 each. Wound size and wound perfusion were assessed and correlated to wound temperature and light absorption in the tissue. Red and trend-wise green light positively stimulated wound healing, while blue light was ineffective. Light absorption was wavelength-dependent and was associated with significantly increased wound perfusion as measured by laser Doppler imaging. Shorter wavelengths ranging from green to blue significantly increased wound surface temperature, while red light, which penetrates deeper into tissue, led to a significant increase in core body temperature. In summary, wound treatment with pulsed red or green light resulted in improved wound healing in diabetic mice. Since impeded wound healing in diabetic patients poses an ever-increasing socio-economic problem, LED therapy may be an effective, easily applied and cost-efficient supportive treatment for diabetic wound therapy.

Effects of Endochondral and Intramembranous Ossification Pathways on Bone Tissue Formation and Vascularization in Human Tissue-Engineered Grafts.

Bone grafts can be engineered by differentiating human mesenchymal stromal cells (MSCs) via the endochondral and intramembranous ossification pathways. We evaluated the effects of each pathway on the properties of engineered bone grafts and their capacity to drive bone regeneration. Bone-marrow-derived MSCs were differentiated on silk scaffolds into either hypertrophic chondrocytes (hyper) or osteoblasts (osteo) over 5 weeks of in vitro cultivation, and were implanted subcutaneously for 12 weeks. The pathways' constructs were evaluated over time with respect to gene expression, composition, histomorphology, microstructure, vascularization and biomechanics. Hypertrophic chondrocytes expressed higher levels of osteogenic genes and deposited significantly more bone mineral and proteins than the osteoblasts. Before implantation, the mineral in the hyper group was less mature than that in the osteo group. Following 12 weeks of implantation, the hyper group had increased mineral density but a similar overall mineral composition compared with the osteo group. The hyper group also displayed significantly more blood vessel infiltration than the osteo group. Both groups contained M2 macrophages, indicating bone regeneration. These data suggest that, similar to the body's repair processes, endochondral pathway might be more advantageous when regenerating large defects, whereas intramembranous ossification could be utilized to guide the tissue formation pattern with a scaffold architecture.

Generation and maturation of human iPSC-derived 3D organotypic cardiac microtissues in long-term culture.

Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs-namely human organotypic cardiac microtissues (hOCMTs)-that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

Bone Graft Packing and Its Association with Bone Regeneration in Maxillary Sinus Floor Augmentations: Histomorphometric Analysis of Human Biopsies.

Research in maxillary sinus floor augmentation (MSFA) focussed on the optimisation of microstructural parameters such as microporosity and particle size of bone substitute particles (BS). However, little is known about the impact of BS packing and the corresponding (void) interparticular space on bone regeneration. The aim of this study was to characterise the spatial distribution of BS and its association with BS integration 6 ± 1 months after MSFA. Histological thin-ground sections of 70 human sinus biopsies were histomorphometrically analysed: In serial zones of 100 µm proceeding from the sinus floor (SF) up to the apical end of the biopsy, we measured the distribution of BS particles within these zones in terms of volume (BSV/TV), number and size of BS particles, interparticle spacing (BS.Sp) and bone-to-BS contact. BS particles were not homogeneously distributed over the length of biopsies: The first 200 µm directly adjacent to the SF represented a zone poor in BS particles but with high osteogenic potential. Graft packing density increased from the SF towards the apical part of the AA. Integration of BS particles was inversely associated with the distance to the SF and the graft packing density. A high packing density through excessive compaction of BS particles should be avoided to optimise the macrostructural environment for bone regeneration.

Tips and Tricks and Clinical Outcome of Cryopreserved Human Amniotic Membrane Application for the Management of Medication-Related Osteonecrosis of the Jaw (MRONJ): A Pilot Study.

Medication-related osteonecrosis of the jaw (MRONJ) is a complication of certain pharmacological treatments such as bisphosphonates, denosumab, and angiogenesis inhibitors. There are currently no guidelines on its management, particularly in advanced stages. The human amniotic membrane (hAM) has low immunogenicity and exerts anti-inflammatory, antifibrotic, antimicrobial, antiviral, and analgesic effects. It is a source of stem cells and growth factors promoting tissue regeneration. hAM acts as an anatomical barrier with suitable mechanical properties (permeability, stability, elasticity, flexibility, and resorbability) to prevent the proliferation of fibrous tissue and promote early neovascularization at the surgical site. In oral surgery, hAM stimulates healing and facilitates the proliferation and differentiation of epithelial cells in the oral mucosa and therefore its regeneration. We proposed using cryopreserved hAM to eight patients suffering from cancer (11 lesions) with stage 2-3 MRONJ on a compassionate use basis. A collagen sponge was added in some cases to facilitate hAM grafting. One or three hAMs were applied and one patient had a reapplication. Three patients had complete closure of the surgical site with proper epithelialization at 2 weeks, and two of them maintained it until the last follow-up. At 1 week after surgery, three patients had partial wound dehiscence with partial healing 3 months later and two patients had complete wound dehiscence. hAM reapplication led to complete healing. All patients remained asymptomatic with excellent immediate significant pain relief, no infections, and a truly positive impact on the patients' quality of life. No adverse events occurred. At 6 months of follow-up, 80% of lesions had complete or partial wound healing (30 and 50%, respectively), while 62.5% of patients were in stage 3. Radiological evaluations found that 85.7% of patients had stable bone lesions (n = 5) or new bone formation (n = 1). One patient had a worsening MRONJ but remained asymptomatic. One patient did not attend his follow-up radiological examination. For the first time, this prospective pilot study extensively illustrates both the handling and surgical application of hAM in MRONJ, its possible association with a collagen sponge scaffold, its outcome at the site, the application of multiple hAM patches at the same time, and its reapplication.

Improved biomechanics in experimental chronic rotator cuff repair after shockwaves is not reflected by bone microarchitecture.

PURPOSE: The aim of this study was to investigate the effect of extracorporeal shockwave therapy (ESWT) on bone microstructure as well as the bone-tendon-interface and the musculo-tendinous transition zone to explain the previously shown improved biomechanics in a degenerative rotator cuff tear animal model. This study hypothesized that biomechanical improvements related to ESWT are a result of improved bone microstructure and muscle tendon properties. METHODS: In this controlled laboratory study unilateral supraspinatus (SSP) tendon detachment was performed in 48 male Sprague-Dawley rats. After a degeneration period of three weeks, SSP tendon was reconstructed transosseously. Rats were randomly assigned into three groups (n = 16 per group): control (noSW); intraoperative shockwave treatment (IntraSW); intra- and postoperative shockwave treatment (IntraPostSW). Eight weeks after SSP repair, all rats were sacrificed and underwent bone microstructure analysis as well as histological and immunohistochemical analyses. RESULTS: With exception of cortical porosity at the tendon area, bone microstructure analyses revealed no significant differences between the three study groups regarding cortical and trabecular bone parameters. Cortical Porosity at the Tendon Area was lowest in the IntraPostSW (p≤0.05) group. Histological analyses showed well-regenerated muscle and tendon structures in all groups. Immunohistochemistry detected augmented angiogenesis at the musculo-tendinous transition zone in both shockwave groups indicated by CD31 positive stained blood vessels. CONCLUSION: In conclusion, bone microarchitecture changes are not responsible for previously described improved biomechanical results after shockwave treatment in rotator cuff repair in rodents. Immunohistochemical analysis showed neovascularization at the musculo-tendinous transition zone within ESWT-treated animals. Further studies focusing on neovascularization at the musculo-tendinous transition zone are necessary to explain the enhanced biomechanical and functional properties observed previously. CLINICAL RELEVANCE: In patients treated with a double-row SSP tendon repair, an improvement in healing through ESWT, especially in this area, could prevent a failure of the medial row, which is considered a constantly observed tear pattern.

Novel Human Placenta-Based Extract for Vascularization Strategies in Tissue Engineering.

There is critical unmet need for new vascularized tissues to support or replace injured tissues and organs. Various synthetic and natural materials were already established for use of two-dimensional (2D) and three-dimensional (3D) in vitro neovascularization assays, however, they still cannot mimic the complex functions of the sum of the extracellular matrix (ECM) in native intact tissue. Currently, this issue is only addressed by artificial products such as Matrigel™, which comprises a complex mixture of ECM proteins, extracted from animal tumor tissue. Despite its outstanding bioactivity, the isolation from tumor tissue hinders its translation into clinical applications. Since nonhuman ECM proteins may cause immune reactions, as are frequently observed in clinical trials, human ECM proteins represent the best option when aiming for clinical applications. Here, we describe an effective method of isolating a human placenta substrate (hpS) that induces the spontaneous formation of an interconnected network of green fluorescence-labeled human umbilical vein endothelial cells (gfpHUVECs) in vitro. The substrate was biochemically characterized by using a combination of bicinchoninic acid (BCA) assay, DNA, and glycosaminoglycan (GAG) content assays, sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE) analysis and Western blot, angiogenesis arrays, chromatographic thrombin detection, high performance liquid chromatography (HPLC)-based amino acid quantification analysis, and assessment of antimicrobial properties. 2D in vitro cell culture experiments have been performed to determine the vasculogenic potential of hpS, which demonstrated that cell networks developed on hpS show a significantly higher degree of complexity (number of tubules/junctions; total/mean tube length) when compared with Matrigel. As 3D cell culture techniques represent a more accurate representation of the in vivo condition, the substrate was 3D solidified using various natural polymers. 3D in vitro vasculogenesis assays have been performed by seeding gfpHUVECs in an hpS-fibrinogen clot. In conclusion, hpS provides a potent human/material-based alternative to xenogenic-material-based biomaterials for vascularization strategies in tissue engineering.

Lugol's solution but not formaldehyde affects bone microstructure and bone mineral density parameters at the insertion site of the rotator cuff in rats.

BACKGROUND: This study aimed to investigate whether rodent shoulder specimens fixed in formaldehyde for histological and histomorphometric investigations and specimens stained using Lugol's solution for soft tissue visualization by micro-computed tomography (microCT) are still eligible to be used for bone architecture analysis by microCT. METHODS: In this controlled laboratory study, 11 male Sprague-Dawley rats were used. After sacrifice and exarticulation both shoulders of healthy rats were assigned into three groups: (A) control group (n = 2); (B) formaldehyde group (n = 4); (C) Lugol group (n = 5). Half of the specimens of groups B and C were placed in a 4% buffered formaldehyde or Lugol's solution for 24 h, whereas the contralateral sides and all specimens of group A were stored without any additives. MicroCT of both sides performed in all specimens focused on bone mineral density (BMD) and bone microstructure parameters. RESULTS: BMD measurements revealed higher values in specimens after placement in Lugol's solution (p < 0.05). Bone microstructure analyses showed increased BV/TV and Tb.Th values in group C (p < 0.05). Specimens of group C resulted in clearly decreased Tb.Sp values (p < 0.05) in comparison to the control group. Formaldehyde fixation showed minimally altered BMD and bone microstructure measurements without reaching any significance. CONCLUSIONS: MicroCT scans of bone structures are recommended to be conducted natively and immediately after euthanizing rats. MicroCT scans of formaldehyde-fixed specimens must be performed with caution due to a possible slight shift of absolute values of BMD and bone microstructure. Bone analysis of specimens stained by Lugol's solution cannot be recommended.

Cre mRNA Is Not Transferred by EVs from Endothelial and Adipose-Derived Stromal/Stem Cells during Vascular Network Formation.

Coculture systems employing adipose tissue-derived mesenchymal stromal/stem cells (ASC) and endothelial cells (EC) represent a widely used technique to model vascularization. Within this system, cell-cell communication is crucial for the achievement of functional vascular network formation. Extracellular vesicles (EVs) have recently emerged as key players in cell communication by transferring bioactive molecules between cells. In this study we aimed to address the role of EVs in ASC/EC cocultures by discriminating between cells, which have received functional EV cargo from cells that have not. Therefore, we employed the Cre-loxP system, which is based on donor cells expressing the Cre recombinase, whose mRNA was previously shown to be packaged into EVs and reporter cells containing a construct of floxed dsRed upstream of the eGFP coding sequence. The evaluation of Cre induced color switch in the reporter system via EVs indicated that there is no EV-mediated RNA transmission either between EC themselves or EC and ASC. However, since Cre mRNA was not found present in EVs, it remains unclear if Cre mRNA is generally not packaged into EVs or if EVs are not taken up by the utilized cell types. Our data indicate that this technique may not be applicable to evaluate EV-mediated cell-to-cell communication in an in vitro setting using EC and ASC. Further investigations will require a functional system showing efficient and specific loading of Cre mRNA or protein into EVs.

Longitudinal Changes of Circulating miRNAs During Bisphosphonate and Teriparatide Treatment in an Animal Model of Postmenopausal Osteoporosis.

MicroRNAs regulate bone homeostasis, and circulating microRNAs have been proposed as novel bone biomarkers. The effect of anti-osteoporotic treatment on circulating microRNAs has not been described in detail. Therefore, we performed a comprehensive analysis of microRNA serum levels in ovariectomized (OVX) and sham-operated (SHAM) rats over 12 weeks of antiresorptive or osteoanabolic treatment. Forty-two Sprague Dawley rats underwent SHAM surgery (n = 10) or ovariectomy (n = 32). After 8 weeks, OVX rats were randomized to antiresorptive treatment with zoledronate (n = 11), osteoanabolic treatment with teriparatide (n = 11), or vehicle treatment (n = 10). Serum samples were collected at weeks 8, 12, 16, and 20 after surgery. A total of 91 microRNAs were analyzed by RT-qPCR in serum samples collected at week 20. Based on the results, 29 microRNAs were selected for longitudinal analysis at all four study time points. Changes in bone mineral density and microstructure were followed up by in vivo micro-CT and ex vivo nano-CT. Ovariectomy resulted in the loss of trabecular bone, which was reversed by osteoanabolic and antiresorptive treatment. Differential expression analysis identified 11 circulating miRNAs that were significantly regulated after treatment. For example, miR-107 and miR-31-5p increased in vehicle-treated OVX animals, whereas they decreased during teriparatide treatment. Additional miRNAs were identified that showed significant correlations to bone microstructure or bone miRNA expression, including miR-203a-3p, which exhibited a significant negative correlation to vertebral and tibial trabecular bone volume fraction (%). Longitudinal analysis confirmed eight microRNAs with significant changes in serum over time that were prevented by teriparatide and zoledronate treatment (miR-34a-5p, miR-31-5p, miR-30d-3p, miR-378a-5p) or teriparatide treatment only (miR-375-3p, miR-183-5p, miR-203a-3p, miR-203b-3p). Gene target network analysis identified WNT and Notch signaling as the main signaling pathways controlled by these miRNAs. Thus, ovariectomy results in time-dependent deregulation of circulating miRNAs compared with SHAM animals. Anti-osteoporotic treatments can rescue this effect, showing that bone-related miRNAs might act as novel biomarkers for treatment monitoring. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Enhanced BMP-2-Mediated Bone Repair Using an Anisotropic Silk Fibroin Scaffold Coated with Bone-like Apatite.

The repair of large bone defects remains challenging and often requires graft material due to limited availability of autologous bone. In clinical settings, collagen sponges loaded with excessive amounts of bone morphogenetic protein 2 (rhBMP-2) are occasionally used for the treatment of bone non-unions, increasing the risk of adverse events. Therefore, strategies to reduce rhBMP-2 dosage are desirable. Silk scaffolds show great promise due to their favorable biocompatibility and their utility for various biofabrication methods. For this study, we generated silk scaffolds with axially aligned pores, which were subsequently treated with 10× simulated body fluid (SBF) to generate an apatitic calcium phosphate coating. Using a rat femoral critical sized defect model (CSD) we evaluated if the resulting scaffold allows the reduction of BMP-2 dosage to promote efficient bone repair by providing appropriate guidance cues. Highly porous, anisotropic silk scaffolds were produced, demonstrating good cytocompatibility in vitro and treatment with 10× SBF resulted in efficient surface coating. In vivo, the coated silk scaffolds loaded with a low dose of rhBMP-2 demonstrated significantly improved bone regeneration when compared to the unmineralized scaffold. Overall, our findings show that this simple and cost-efficient technique yields scaffolds that enhance rhBMP-2 mediated bone healing.

The vertical course of bone regeneration in maxillary sinus floor augmentations: A histomorphometric analysis of human biopsies.

BACKGROUND: Maxillary sinus floor augmentation (MSFA) is a well-established and predictable augmentation method in severely resorbed maxillae. However, data on the vertical course of bone graft consolidation within the maxillary sinus are rare. The aim of the present study was to quantify the vertical distribution of new bone formation (nBF) in MSFA and to characterize the vertical gradient of bone graft consolidation. METHODS: Eighty-five human sinus biopsies were harvested 6 ± 1 months after MSFA. Histological thin-ground sections were prepared and histomorphometrically analyzed. The volume of newly formed bone (nBV/TV) was measured in serial zones of 100 µm proceeding from the bottom of the sinus floor (SF) up to the apical top of the biopsy. The gradient of nBV/TV within the augmentation area was determined by the vertical distribution of nBV/TV along these zones. RESULTS: In the premolar region, nBV/TV slightly declined from 20.4% in the zone adjacent to the SF to 17.7% at a distance of 8 mm. The gradient was steeper in the molar region: nBV/TV decreased from 18.7% to 12.8%. This decline was even more distinct when the volume fraction and the height of the residual bone of the SF were low. CONCLUSIONS: nBF follows a gradient from native bone of the SF towards the apical part of the augmentation area. The distance to primordial bone thus plays a critical role for bone regeneration in MSFA, particularly in the molar region.

A novel fluorescent hydroxyapatite based on iron quantum cluster template to enhance osteogenic differentiation.

Template-mediated self-assembly synthesis has produced a diverse range of biomimetic materials with unique physicochemical properties. Here, we fabricated novel fluorescent three-dimensional (3-D) hydroxyapatite (HAP) nanorod-assembled microspheres using iron quantum cluster (FeQC) as a hybrid template, containing three organic components: hemoglobin chains, piperidine, and iron clusters. The material characterization indicated that the synthesized HAP possessed a uniform rod-like morphology, ordered 3-D architecture, high crystallinity, self-activated fluorescence, and remarkable photostability. Our study proposed that this FeQC template is a promising regulating agent to fabricate fluorescent self-assembled HAP microspheres with a controlled morphology. The effect of HAP on stem cell fate and their osteogenic differentiation was investigated by culturing human bone marrow-derived mesenchymal stromal/stem cells (BMSCs) with HAP microspheres. Significant increases in collagen matrix production and gene expression of osteogenic markers, including osteocalcin (OCN), Runt-related transcription factor 2 (Runx2), bone sialoprotein (BSP) and alkaline phosphatase (ALP), were observed compared to the controls after 21 days of culture. Taken together, our data suggest that synthetic HAP nanorod-assembled microspheres represent a promising new biomaterial which exhibits enhanced fluorescent properties and osteoinductive effects on human BMSCs.

The course of recovery of locomotor function over a 10-week observation period in a rat model of femoral nerve resection and autograft repair.

BACKGROUND: A great extent of knowledge on peripheral nerve regeneration has been gathered using the rat sciatic nerve model. The femoral nerve model of the rat offers an interesting alternative, as it lacks disadvantageous features such as automutilation. For the analysis of locomotor behavior in rats after sciatic nerve injury, the CatWalk™ XT Gait Analysis System is often used. However, lesions of the femoral nerve in the rat have yet remained unstudied with this method. MATERIAL AND METHODS: Ten male Sprague Dawley rats were evaluated with the CatWalk XT to study their gait after a 6-mm resection of the right femoral nerve and reconstruction with an autologous nerve graft. Animals were observed for 10 weeks after surgery. RESULTS: Print Area, Print Length, Swing Speed, and Duty Cycle decreased to a minimum of 40% of baseline 2 weeks after surgery. Swing Time was elevated more than twofold at this time point. However, all these parameters recovered back to >90% of baseline values at 10 weeks after surgery. This degree of functional recovery has not been reported after sciatic nerve resection and autograft repair. Base of support varied minimally postoperatively in contrast to a strong decrement after sciatic nerve resection and repair. CONCLUSION: We hereby provide a comprehensive in-depth analysis of how to study functional recovery after injury of the femoral nerve in the rat via the CatWalk XT. We place special emphasis on highlighting the differences between the femoral nerve and sciatic nerve injury model in this context.

Purinergic P2Y2 receptors modulate endothelial sprouting.

Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.

An Efficacy Comparison of Two Hemostatic Agents in a Porcine Liver Bleeding Model: Gelatin/Thrombin Flowable Matrix versus Collagen/Thrombin Powder.

Purpose: Management of bleeding during surgery can be aided by the application of topical hemostatic agents. This study compared the hemostatic efficacy of a new powder agent containing collagen, chondroitin sulfate, and thrombin (PCCT) with a flowable gelatin-thrombin matrix with smooth particles (SmGM) in a porcine liver bleeding model. Materials and Methods: Lesions 4-6 mm deep and ∼10 mm in diameter were created in porcine livers and treated with either SmGM or PCCT. Bleeding rate and grade were quantified before and 3, 7, and 11 minutes after treatment. Results: Thirty-two lesions each were treated with SmGM or PCCT; the median (Q1, Q3) initial bleeding rate was comparable between the two groups (8.43 [6.18, 10.68] g/min and 7.15 [5.16, 9.63] g/min, respectively). The residual bleeding rate was significantly lower at all time-points post treatment for SmGM compared with PCCT (3 minutes: 0.14 [0.07, 0.21] versus 0.46 [0.20, 1.20] g/min, p < 0.0001; 7 minutes: 0.07 [0.04, 0.11] versus 0.12 [0.08, 0.39] g/min, p = 0.001; 11 minutes: 0.05 [0.03, 0.08] versus 0.07 [0.05, 0.12] g/min, p = 0.043). Bleeding grade at 3 minutes was also significantly lower for SmGM compared with PCCT (median [Q1, Q3] 0.0 [0.0, 0.0] versus 1.0 [1.0, 2.0], p < 0.0001). PCCT required reapplication in approximately one-third of applications due to insufficient hemostasis 4 minutes after initial application and showed a tendency to stick to the wet gauze during approximation. Conclusions: In this bleeding model, treatment with SmGM resulted in reduced blood loss, no need for reapplication and was easier to apply compared with PCCT.

MicroRNA levels in bone and blood change during bisphosphonate and teriparatide therapy in an animal model of postmenopausal osteoporosis.

MicroRNAs control the activity of a variety of genes that are pivotal to bone metabolism. Therefore, the clinical utility of miRNAs as biomarkers and drug targets for bone diseases certainly merits further investigation. This study describes the use of an animal model of postmenopausal osteoporosis to generate a comprehensive dataset on miRNA regulation in bone tissue and peripheral blood during bone loss and specifically anti-resorptive and osteo-anabolic treatment. Forty-two Sprague-Dawley rats were randomized to SHAM surgery (n=10) or ovariectomy (OVX, n=32). Eight weeks after surgery, OVX animals were further randomized to anti-resorptive treatment with zoledronate (n=11), osteo-anabolic treatment with teriparatide (n=11), or vehicle treatment (n=10). After 12 weeks of treatment, bone and serum samples were used for microRNA analysis using next-generation sequencing (NGS), mRNA levels using RT-qPCR, and bone microarchitecture analysis using nanoCT. Ovariectomy resulted in loss of trabecular bone, which was fully rescued using osteo-anabolic treatment, and partially rescued using anti-resorptive treatment. NGS revealed that both, anti-resorptive and anabolic treatment had a significant impact on miRNA levels in bone tissue and serum: out of 426 detected miRNAs, 46 miRNAs were regulated by teriparatide treatment an d 10 by zoledronate treatment (p-adj.<0.1). Interestingly, teriparatide and zoledronate treatment were able to revert miRNA changes in tissue and serum of untreated OVX animals, such as the up-regulation of miR-203a-3p, a known osteo-inhibitory miRNA. We confirmed previously established mechanisms of miR-203a by analyzing its direct target Dlx5 in femoral head. Our data reveal a significant effect of ovariectomy-induced bone loss, as well as the two major types of anti-osteoporotic treatment on miRNA transcription in femoral head tissue. These changes are associated with altered activity of target genes relevant to bone formation, such as Dlx5. The observed effects of bone loss and treatment response on miRNA levels in bone are also reflected in the peripheral blood, suggesting the possibility of minimally-invasive monitoring of bone-derived miRNAs using liquid biopsies.