Guided bone regeneration (GBR) relies on barrier membrane integrity to prevent soft-tissue ingrowth. Although collagen membranes are widely used, their limited longevity can compromise space maintenance, underscoring the need for strategies that enhance membrane stability without impairing the regenerative potential. We hypothesized that thermal denaturation of platelet-poor plasma (PPP), combined with heat-induced modifications of collagen fibrils, could generate a volume-stable, plasma-rich composite that preserves membrane structure and restricts cellular penetration. To test this proof-of-principle concept, collagen membranes were soaked in PPP and either kept at room temperature or subjected to thermal treatment (75 °C/10 min) prior to implantation in rat calvarial defects. Bone regeneration and membrane behavior were evaluated after three weeks using micro-computed tomography (micro-CT) and histology. Micro-CT suggested only minor numerical differences in mineralized tissue between groups; however, these data should not be overinterpreted because micro-CT cannot differentiate mineralization formed within the collagen membrane from mineralization adjacent to it. Consistent with this limitation, histology demonstrated that mineral deposition and early bone formation extended into the structure of room-temperature PPP membranes, whereas mineralized tissue in the thermally treated group was predominantly located outside the membrane, indicating reduced osteoconductive integration within the membrane. Together, these findings support that thermal denaturation of PPP shifts early composite membrane behavior toward barrier-dominant characteristics at the expense of intramembranous mineralization.

OBJECTIVE:To evaluate the efficacy of mesenchymal stromal cells' (MSC) secretome delivered as conditioned media (CM) as an adjunct to a xenograft (XG) and collagen membrane (MEM) for guided bone regeneration (GBR) in chronic non-contained mandibular defects in minipigs. METHODS:Chronic bilateral mandibular defects were surgically created in seven minipigs. In a split-mouth design, defects were treated with either CM-functionalized (test) or native XG + MEM (control) and evaluated after 4 and 12 weeks. New bone formation was assessed using in vivo computed tomography (CT) and ex vivo micro-CT, histology and immunohistochemistry (IHC). Quantitative CT, micro-CT and histomorphometric data were statistically analysed. RESULTS:No significant differences in new bone formation were observed between the test and control group at either timepoint. These findings could be partly explained by a high intrinsic healing capacity and continual growth of the animals. Minimal residual XG material was detected in both groups at 4 and 12 weeks. When present, these remnants were associated with multinucleated Cathepsin K-positive cells (IHC) with no observable differences between the groups. CONCLUSION:The adjunctive use of MSC secretome in combination with XG and MEM did not improve GBR outcomes in chronic non-contained mandibular defects in the present minipig model. The suitability of this animal model for experimental GBR may be questionable.

The process of endochondral ossification (EO) is not only involved in the foetal development of long bones but was employed for bone tissue engineering. Its success was confirmed in multiple animal models showing that chondrogenic templates can develop into bone upon implantation in vivo. Nevertheless, the utilization of EO for regeneration of osteochondral tissues remains challenging due to limitations of stable articular cartilage formation. Here, we report a synergetic bottom-up tissue engineering approach, based on scaffolded spheroids (SSPH) obtained by combining high-resolution 3D printed microscaffolds with chondrogenic primed spheroids produced from mesenchymal stem cells (MSC). This approach allows the bioassembly of millimetre-sized tissue constructs with control over differentiation of individuals as reported previously for cartilage-like tissue. Here, we show the successful regeneration of critical-sized osteochondral defects in vivo in rabbits. The influence of vascularization is discussed in the context of bone regeneration, suggesting a direct influence of increased vascularization for improved regeneration outcomes. The cartilage regeneration results show a significantly better regeneration for treated defect sites compared to empty controls. Cartilage resurfaced, and gave rise to nearly full thickness cartilage-like tissue, and full integration into the surrounding tissue. The present study provides a proof of concept for using S-SPHs for cartilage repair in osteochondral defects in rabbit and shows the potential for bone regeneration in vivo.

AIM:To assess the effect of a hyaluronic acid (HyA) containing gel on patient-related outcomes (PRO) and wound healing after palatal punch-biopsy representing a free gingival graft. MATERIAL AND METHODS:A punch-biopsy (6 mm diameter, 2 mm thickness) was harvested from one side of the palate at Day 0 and from the contralateral side at Day 21. Sites were randomly allocated to either 0.3% HyA containing gel (test) or sterile saline solution (control), professionally applied after harvesting and then self-applied three times/day for 7 days. Up to 21 days PRO-related questionnaires were answered and intraoral scans and photographs recorded to assess wound re-epithelialization, tissue refill, and color match. RESULTS:Eighteen of 25 recruited participants were analyzed. Pain perception, difficulties with eating/drinking, and taste alterations significantly decreased over time and were hardly experienced after Day 7 in both groups (p > 0.05). Participants reported significantly more often a positive experience after applying HyA compared to placebo, that is, in 8 versus 1 out of 18 cases. The residual wound area was significantly smaller in the test compared to control group at Day 7, and cases with a thicker palatal tissue showed tendency for faster re-epithelialization. CONCLUSION:Despite the potential limitations of the present study (i.e., possibility of a carryover effect, questionable blinding of the participants), repeated local application of a 0.3% HyA containing gel appears to only accelerate re-epithelialization in an open palatal wound and provide a positive experience after application, but it did not improve any PRO in such small-sized wounds compared to sterile saline application. TRIAL REGISTRATION:ClinicalTrials.gov identifier: NCT05099718.

AIM:To determine whether myeloid-specific deletion of A20 (TNFAIP3), a key negative regulator of NF-κB signaling, affects periodontal supporting tissues and temporomandibular joint integrity under baseline conditions. METHODS:A20myel-KO and wild-type littermates were analyzed at 10-11 weeks of age using high-resolution micro-computed tomography and histology. Alveolar bone architecture, periodontal ligament space, epithelial morphology, and temporomandibular joint (TMJ) compartments were evaluated through quantitative micro-CT measurements and qualitative histological assessment. RESULTS:A20myel-KO mice exhibited a craniofacial structural phenotype characterized by reduced alveolar bone volume fraction, widening of the periodontal ligament space, and altered dentoalveolar morphology. Histological analysis revealed epithelial architectural changes. In the TMJ, micro-CT demonstrated reduced bone volume fraction, accompanied by histological evidence of trabecular bone loss and altered condylar growth plate organization. CONCLUSION:A20-mediated regulation in myeloid cells contributes to the maintenance of periodontal and craniofacial skeletal homeostasis. Loss of this regulatory pathway was associated with structural alterations involving alveolar bone loss, periodontal ligament architecture, and temporomandibular joint morphology, suggesting that dysregulated myeloid inflammatory signaling may influence multiple craniofacial skeletal compartments even in the absence of experimentally induced inflammation.

Introduction Bone healing is a well-orchestrated process involving various bone cells and signaling pathways, where disruptions can result in delayed or incomplete healing. MicroRNAs (miRNAs) are small non-coding RNAs capable of influencing various cellular processes, including bone remodeling. Due to their biological relevance and stable presence in biofluids, miRNAs may serve as candidates for diagnosis and prognosis of delayed bone healing. The aim of the study was to investigate changes in miRNAs circulating in the blood during the healing of rat calvaria defects as biomarkers of successful bone regeneration.Methods Standardized calvaria defects were created in 36 Wistar rats with a trephine drill and treated with collagen hydroxyapatite (CHA) scaffolds. The treatment groups included CHA scaffolds only, CHA scaffolds containing a plasmid coding for bone morphogenetic protein 2 (BMP2) and miR-590-5p, CHA scaffolds containing mesenchymal stromal cell-derived extracellular vesicles, and empty defects as a control group. After 1, 4 and 8 weeks of healing, the animals were evaluated by microcomputed tomography (microCT), as well as subjected to histological analyses. Blood was sampled from the tail vein prior to surgeries and after 1, 4, and 8 weeks of healing. miRNAs circulating in the plasma were determined using next-generation sequencing.Results Variability of bone regeneration within the four groups was unexpectedly high and did not result in significant differences between the groups, as indicated by the microCT and histological analyses of the newly formed bone tissue. However, irrespective of the treatment group and regenerative activity, we identified miRNAs with distinct expression patterns of up- and downregulation at different time points. Furthermore, rats with high and low regenerative activity were characterized by distinct circulating miRNA profiles. miR-133-3p was identified as the top upregulated miRNA and miR-375-3p was identified as the top downregulated miRNA in animals exhibiting strong regeneration over all time points evaluated.Conclusion Our study indicates that regardless of the treatment group, success or lack of bone regeneration is associated with a distinct expression pattern of circulating microRNAs. Further research is needed to determine whether their levels in the blood can be used as predictive factors of successful bone regeneration.

The aim of this study was to grow axially vascularized soft tissue flaps in sheep using the arteriovenous loop (AVL) technique to be transplanted for defect reconstruction. This technique may be a promising alternative to conventional free flaps to further reduce flap donor site morbidity. In this pilot study, AVLs (n = 12) were created in the groins of six sheep, placed into an isolation chamber, and embedded in Matriderm®. Tissue volume, vascularization, and cell proliferation were assessed on postoperative day (POD) 28 using immunohistochemical staining and microcomputed tomography (µCT). Four AVL free flaps were microsurgically anastomosed to the neck vessels in a standardized defect sheep model on POD 28. Defect closure and intrinsically vascularized scaffold-based bioengineered flaps (IVSBs) flap perfusion were studied by angiography and histology 10 days after transplantation. One IVSB flap was lost due to chamber infection. At POD 28, the remaining 11 IVSB flaps had filled the isolation chamber. Histological examination and µCT analysis of seven IVSB flaps verified homogeneous microvascular networks within the flaps. The mean number of microvessels, vessel volume, and the percentage of proliferating cells increased significantly over time. In the defect model, all four transplanted flaps showed macroscopically, angiographically, and histologically stable defect closure 10 days after transplantation, with homogeneous vascular integration into the surrounding tissue. This pilot study demonstrates that in a large animal model complex, defects can be reconstructed using free IVSB flaps with a clinically relevant tissue volume. These data provide the preclinical proof prior to human application.

Decellularized articular cartilage of human origin presents itself as the most homologous filling material for focal cartilage defects. Yet, the full repopulation of the exceptionally dense collagen construct has never been achieved without providing host cells with artificially created migration paths into the matrix. Within this study, we examine the use of a femtosecond laser to engrave fine patterns into human articular cartilage before decellularization and GAG depletion (decell-deGAG). Scaffolds were tested for decellularization success and mechanical behavior. Seeding tests were performed to assess biocompatibility and examine the performance in a simulated defect environment using an osteochondral plug model in vitro and in vivo in an ectopic nude mouse model. The composition and structure of the newly formed repair tissue and macrophage recruitment were observed via histology. The femtosecond laser was successful in engraving deep, fine structures into the matrix without the thermal damage found with other laser techniques. Engraving was also beneficial for decellularization success. The resulting decell-deGAG scaffold featured a compressive modulus many times stronger than other biomaterials commonly used for cartilage regeneration and presents a defect filling material that is similar to the tissue it is meant to replace. Moreover, the incisions promoted the repopulation with therapeutically relevant cells. A favorable spatial environment inside the incisions facilitated the formation of repair tissue that mimics hyaline cartilage in composition and collagen orientation. Scaffolds were well-integrated within simulated defects. Femtosecond laser-engraved cartilage poses an authentic defect filling material with cartilage-like properties. When used in combination with cell seeding, it promotes the formation of differentiated repair tissue. Thus, the hereby presented biomaterial shows great potential in improving the repair of focal cartilage defects and reducing long-term graft failures.

OBJECTIVE:To evaluate, in a simulation of surgical peri-implantitis treatment, the impact of type of handpiece, device settings, and instrumentation time on the efficacy of airflowing in cleaning the implant surface, depending on the type of bone defect and implant surface. METHODS:Turned and modified surface implants (54 each) were coated with biofilm imitation and mounted on resin models replicating purely horizontal or circumferential intraosseous peri-implant defects (both 5 mm deep). Implants were instrumented with an airflowing device using a supra- or submucosal handpiece, with three settings: (a) power 5, 5 s (b) power 10, 5 s, and (c) power 5, 15 s per implant/defect sextant. RESULTS:The amount of residual biofilm imitation was associated with defect configuration, type of handpiece, and device settings (p < 0.15); implant surface did not have an effect. In horizontal defects, with the supramucosal handpiece, only 3 of 54 implants showed > 5% residual biofilm imitation and 23 of 54 implants were completely clean; with the submucosal handpiece, 12 of 18 implants showed ≤ 5% residual biofilm imitation when used for 15 s/sextant, yet none were completely clean. In intraosseous defects, all implants presented ≤ 5% residual biofilm imitation and 10 of 18 implants were completely clean with the submucosal handpiece used for 15 s/sextant; the supramucosal handpiece was largely inefficacious. CONCLUSION:Within the limitations of this laboratory study, peri-implant bone defect configuration should dictate the choice of airflowing handpiece (i.e., for horizontal defects, the supramucosal handpiece; for intraosseous defects, the submucosal handpiece) and intrasurgical airflowing requires a prolonged instrumentation time, but not increased power.

OBJECTIVE:To evaluate the impact of implant surface and instrumentation time on the efficacy of two air powder water-jet (APWJ) devices in cleaning the implant surface in a simulation of non-surgical peri-implantitis treatment. MATERIALS AND METHODS:Turned and modified surface implants (28 each) were coated with a biofilm imitation and mounted on resin models replicating peri-implant intra-osseous defects, including a soft tissue replica. The entire implant periphery was instrumented for 5 or 15s per implant sextant (i.e., in total 30 or 90s per implant), with one of two different APWJ devices using either a glycine or an erythritol powder. Residual biofilm imitation was automatically assessed on standardized photographs and expressed as percentage of the exposed implant surface. RESULTS:Implant surface (ε2:0.253, p < 0.001) and instrumentation time (ε2:0.044, p = 0.036) had a moderate and small effect, respectively, on the outcome, that is, instrumenting turned compared to modified surface implants as well as using a longer compared to a shorter instrumentation time resulted in less residual biofilm imitation. Complete biofilm imitation removal was achieved only in four turned implants, treated for 15s per sextant. Every second turned implant presented with a maximum of 5% residual biofilm imitation, while only two modified implants achieved this level of cleanliness. CONCLUSION:In a non-surgical peri-implantitis treatment simulation with APWJ devices, superior biofilm imitation removal was achieved at turned implants, and a longer instrumentation time resulted in less residual biofilm imitation. Modified implants had high chances of incomplete biofilm imitation removal, especially at the apical part of the defect. Complete biofilm imitation removal was in general largely unpredictable.

Introduction Tooth mineralisation and alveolar eruption show a strong temporal correlation with the chronological age of sub-adults and thus represent a valuable asset for age estimation in archaeology and forensic science. Despite the vast number of various dental age estimation methods published, evidence-based comparison of their degree of accuracy is lacking. This study aimed (1) to evaluate if mu CT scans are a reliable alternative to conventional techniques for tooth length measurements and (2) to compare the accuracy and reliability of the London Atlas of Tooth Development and Eruption with six well-established regression methods for age estimation of subadults. Material and Methods Forty-two skulls of a Central European osteological collection of the early 19th century (known age at death 7 months in utero-11 years) were scanned with mu CT to estimate dental age via London Atlas-classification and regression equations based on deciduous and permanent tooth length measurements. Results Tooth length measurements via mu CT proved to be a technically feasible method with an excellent agreement with caliper measurements.Regression equations of Liversidge (1993) and Cardoso (2019) for the deciduous dentition and London atlas yielded the most accurate age estimates (prediction errors: 0.20-0.30 years). Regression equations for permanent teeth were less accurate; however, performance was improved when their application was restricted to individuals <= 3 years of age. Most accurate age estimates for single teeth were achieved for deciduous incisors, canines and first molars. Discussion mu CT extends the spectrum of tools available for tooth length assessment. While regression equations for deciduous teeth represent a reliable age estimation method for younger individuals, the London Atlas outperforms the regression equations for permanent teeth. It thus can be considered a preferred alternative to estimate age in older individuals (>3 years) with an adequate state of conservation. A higher weighting of the first mineralising deciduous teeth can contribute to further improving the accuracy of the London Atlas.

Traumatic spinal cord injury is a life changing condition, and treatment options are still limited. While a great deal of work has been done in the last decade to improve the understanding of tissue degeneration and repair, more work is necessary to translate this knowledge into the clinic to improve the outcome for patients. Imaging modalities for the spinal cord play an important role in this process but are currently mostly limited to destructive two-dimensional histology and expensive, low-resolution 3D MRI. This work seeks to expand the existing ex vivo imaging possibilities in preclinical research with a cost-effective, non-destructive technique for both healthy and injured spinal cords.We imaged 13 spinal cords using Lugol’s iodine, Hf-POM and Accupaque as contrast enhancing staining agents (CESAs) for µCT imaging. These included: uninjured spinal cords of 8 rats, 4 rat spinal cords with a contusion injury as well as one healthy spinal cord from a human body donor. CECT acquisitions were performed with an array of different settings at resolutions between 1.2 µm for detailed structural evaluation and 18 µm for contusion injury quantification. We further performed comparative classical histological analysis on 5 rat spinal cords and the human spinal cord for validation purposes.For the first time, .we obtained high resolution structural information of the healthy spinal cord using CESAs Lugol’s iodine and Hf-POM, with clear distinction of white and gray matter, visualization of numerous neuronal tracts and fiber bundles. We could detect individual cells such as motoneurons, down to the small fibers of the intramedullary bundles, which run from motoneurons to the ventral rootlets. We could further demonstrate compatibility of immunohistology post-processing with CECT. In the injured spinal cord, both Lugol’s iodine and Accupaque staining provide useful information regarding the extent and composition of the lesioned area.We demonstrate that CECT imaging provides unprecedented 3D structural detail in a non-destructive manner. Though limited to ex-vivo applications, this method provides resolutions that far exceed those achievable with current MRI technology. We provide examples of staining procedures for a range of applications of the spinal cord. We believe the techniques described here are especially useful in combination with classical histological examination and provide additional details in a cost-effective manner.

Spinal cord injury (SCI) is a complex clinical condition with a wide range of permanent functional and neurological consequences. A prime factor limiting the patient’s quality of life (QoL) is difficulties in bladder function. Chronic animal models that help to develop novel therapeutic strategies are highly demanded, but their availability is scarce and frequently accompanied by substantial limitations. We want to provide our detailed protocols that allow full reproducibility of a novel model for investigating both the acute and chronic condition, and give transparency regarding challenges. The preclinical animal model of female rats with mid-thoracic SCI contusion and a permanently implanted urinary catheter allowed the measuring of bladder function repetitively. Over a period of six months, data were collected weekly from the same, conscious individuals. To our knowledge, this is the first study that obtained a clinically relevant urodynamic dataset seamlessly from the acute to the chronic phase in rats with SCI. The ability to generate a complete data set from one single individual, rather than requiring multiple subjects, has the potential to markedly reduce the number of experimental animals, eliminate group differences, and give more flexibility for therapeutic intervention. Future projects could also benefit from the described optimizations in animal care.

This study aims to investigate the impact of the pro-osteoblastogenic ERK-activated ribosomal S6 kinase (Rsk2) on Tumor necrosis factor (TNF)α-induced bone loss in the craniofacial system, focusing on its role in rheumatoid arthritis (RA). The objective is to understand whether Rsk2, previously shown to have protective effects in long bones against TNFα-induced bone resorption, exhibits similar effects in the craniofacial region. , and Methods. The study compares mice with TNFα overexpression, Rsk2 knockout mice, and a combination of TNFα, and Rsk2 knockout mice using detailed micro-computed tomography coupled with landmark based morphometric analysis, and classical histology. The overall skull morphology, mandible shape, and the temporomandibular joint were examined. Additionally, histological sections were utilized to examine the synovial membrane. Combining TNFα, and Rsk2 deficiency does not further alter overall skull shape compared to TNFα alone. TNFα overexpression shortens the mandibular ramus, exacerbated by Rsk2 absence. Micro-computed tomography (µCT) reveals significant temporomandibular joint damage from TNFα, independent of Rsk2. However, histological sections show increased synovial membrane thickness with TNFα, heightened in the absence of Rsk2. Rsk2 mitigates TNFα-induced effects on mandibular ramus length in the craniofacial system but has limited impact on the temporomandibular joint, except for synovial membrane thickness. Overall, Rsk2 demonstrates a weaker preventive effect on TNFα-induced craniofacial bone loss compared to its established role in the appendicular skeleton. This study highlights regional differences in Rsk2's protective mechanisms, emphasizing the need for further exploration of the underlying mechanisms for these disparities. Understanding these regional differences can be crucial for the development of targeted therapeutic interventions.

Abstract Objectives To evaluate in the absence of teeth the variability of the mylohyoid line (ML), the microarchitecture of the adjacent bone, and whether the variable prominence/width of the ML is associated with the quality of the adjacent bone. Methods µCT scans of 28 human mandibles from anatomical specimens were analyzed. The following parameters were assessed in four edentulous areas (first and second premolar (PM), first, second, and third molar (M1/2/3)): ML width, cortical thickness (CtTh), average cortical- (Avg.Ct.BV/TV), and trabecular bone volume fraction (Avg.Tb.BV/TV). Results The ML width increased from the PM towards the M2 region, which also showed the highest variance (range: 0.4–10.2 mm). The CtTh showed a decrease in the M3 region, while Avg.Ct.BV/TV and Avg.Tb.BV/TV hardly differed among the regions. In the multivariable model on the effect of the various parameters on the ML width, only gender and tooth region were significant. Specifically, male specimens were associated with a wider ML width compared to female specimens and the M2 region was associated with a wider ML width compared to the other tooth regions. Conclusion The ML width was not associated with the cortical and trabecular bone quality in the adjacent bone, while gender and tooth region had a significant effect. Specifically, the ML width was lower in female, but peaked in the M2 region with a median width of 3–4 mm. Clinical relevance From a clinical point of view, it was confirmed that the ML is in general a highly variable structure, especially in the M2 region, but the ML width does not allow any conclusions on the bone quality. Altogether, this underlines the need for an individual and accurate diagnostic prior to any surgical intervention.

Abstract 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.

Background: Free flap–based soft-tissue reconstruction comes at the price of donor-site morbidity. The arteriovenous loop (AVL) technique can overcome this issue by allowing for the de novo generation of axially vascularized soft-tissue flaps from vein grafts embedded into different matrices. Application of the AVL technique has been limited by insufficient long-term volume retention and poor tissue stability. The authors investigated the suitability of a novel human dermal scaffold to improve volume retention and tissue stability. Methods: AVLs were created in 28 immunocompetent rats and embedded in either decellularized human dermal scaffolds (experimental group, n = 14) (Epiflex) or bovine collagen/elastin matrices (control group, n = 14) (MatriDerm) in subcutaneous polytetrafluoroethylene chambers. The weight and volume of engineered tissues, the extent of angiogenesis, and the proportion of proliferating cells were compared between groups on postoperative days (PODs) 21 and 28 by means of immunohistochemistry and micro–computed tomography. Results: On POD 28, both groups displayed homogeneous microvascular networks on histopathology and micro–computed tomography. Mean microvessel counts and surface areas and the percentage of proliferating cells did not differ between the groups. However, the experimental human scaffold group displayed significantly smaller volume loss and significantly less tissue degradation compared with bovine matrix controls (volume retention, 102% ± 5% versus 27% ± 7% on POD 21, and 79% ± 12% versus 12% ± 7% on POD 28, respectively; P < 0.0001). Conclusion: Compared with bovine matrices, decellularized human scaffolds allow for superior volume retention and tissue stability of de novo engineered soft-tissue AVL flaps in rats. Clinical Relevance Statement: AVLs allow for the de novo generation of vascularized soft-tissue flaps. However, insufficient long-term volume retention is still an issue. The authors’ study shows that decellularized human matrices guarantee superior volume stability of de novo grown soft-tissue flaps in rats.

Peripheral nerve injuries induce a severe motor and sensory deficit. Since the availability of autologous nerve transplants for nerve repair is very limited, alternative treatment strategies are sought, including the use of tubular nerve guidance conduits（tNGCs）. However, the use of tNGCs results in poor functional recovery and central necrosis of the regenerating tissue, which limits their application to short nerve lesion defects（typically shorter than 3 cm）. Given the importance of vascularization in nerve regeneration, we hypothesized that enabling the growth of blood vessels from the surrounding tissue into the regenerating nerve within the tNGC would help eliminate necrotic processes and lead to improved regeneration. In this study, we reported the application of macroscopic holes into the tubular walls of silk-based tNGCs and compared the various features of these improved silk + tNGCs with the tubes without holes（silk – tNGCs） and autologous nerve transplants in an 8-mm sciatic nerve defect in rats. Using a combination of micro-computed tomography and histological analyses, we were able to prove that the use of silk + tNGCs induced the growth of blood vessels from the adjacent tissue to the intraluminal neovascular formation. A significantly higher number of blood vessels in the silk + group was found compared with autologous nerve transplants and silk – , accompanied by improved axon regeneration at the distal coaptation point compared with the silk – tNGCs at 7 weeks postoperatively. In the 15-mm（critical size） sciatic nerve defect model, we again observed a distinct ingrowth of blood vessels through the tubular walls of silk + tNGCs, but without improved functional recovery at 12 weeks postoperatively. Our data proves that macroporous tNGCs increase the vascular supply of regenerating nerves and facilitate improved axonal regeneration in a short-defect model but not in a critical-size defect model. This study suggests that further optimization of the macroscopic holes silk + tNGC approach containing macroscopic holes might result in improved grafting technology suitable for future clinical use.

Age-induced decline in osteogenic potential of bone marrow mesenchymal stem cells (BMSCs) potentiates osteoporosis and increases the risk for bone fractures. Despite epidemiology studies reporting concurrent development of vascular and bone diseases in the elderly, the underlying mechanisms for the vascular-bone cross-talk in aging are largely unknown. In this study, we show that accelerated endothelial aging deteriorates bone tissue through paracrine repression of Wnt-driven-axis in BMSCs. Here, we utilize physiologically aged mice in conjunction with our transgenic endothelial progeria mouse model (Hutchinson-Gilford progeria syndrome; HGPS) that displays hallmarks of an aged bone marrow vascular niche. We find bone defects associated with diminished BMSC osteogenic differentiation that implicate the existence of angiocrine factors with long-term inhibitory effects. microRNA-transcriptomics of HGPS patient plasma combined with aged-vascular niche analyses in progeria mice reveal abundant secretion of Wnt-repressive microRNA-31-5p. Moreover, we show that inhibition of microRNA-31-5p as well as selective Wnt-activator CHIR99021 boosts the osteogenic potential of BMSCs through de-repression and activation of the Wnt-signaling, respectively. Our results demonstrate that the vascular niche significantly contributes to osteogenesis defects in aging and pave the ground for microRNA-based therapies of bone loss in elderly.

Platelet-rich fibrin, the coagulated plasma fraction of blood, is commonly used to support natural healing in clinical applications. The rat calvaria defect is a standardized model to study bone regeneration. It remains, however, unclear if the rat calvaria defect is appropriate to investigate the impact of human PRF (Platelet-Rich Fibrin) on bone regeneration. To this end, we soaked Bio-Gide® collagen membranes in human or rat liquid concentrated PRF before placing them onto 5 mm calvarial defects in Sprague Dawley rats. Three weeks later, histology and micro-computed tomography (μCT) were performed. We observed that the collagen membranes soaked with rat PRF show the characteristic features of new bone and areas of mineralized collagen matrix, indicated by a median mineralized volume of 1.5 mm3 (range: 0.9; 5.3 mm3). Histology revealed new bone growing underneath the membrane and hybrid bone where collagen fibers are embedded in the new bone. Moreover, areas of passive mineralization were observed. The collagen membranes soaked with human PRF, however, were devoid of histological features of new bone formation in the center of the defect; only occasionally, new bone formed at the defect margins. Human PRF (h-PRF) caused a median bone volume of 0.9 mm3 (range: 0.3–3.3 mm3), which was significantly lower than what was observed with rat PRF (r-PRF), with a BV median of 1.2 mm3 (range: 0.3–5.9 mm3). Our findings indicate that the rat calvaria defect model is suitable for assessing the effects of rat PRF on bone formation, but caution is warranted when extrapolating conclusions regarding the efficacy of human PRF.