Bone regeneration represents still a challenge, in particular for calvarium defects. Recently, the development of biomaterials with the addiction of stem cells is giving promising results for the treatment of bone defects. In particular, it was demonstrated that scaffolds enriched with mesenchymal stem cells (MSCs) and/or their derivatives, such as conditioned medium (CM) and extracellular vesicles (EVs), may improve bone regeneration. Moreover, given the deep link between osteogenesis and angiogenesis, a successful approach must also take into consideration the development of vascularization. In this work we evaluated the bone regeneration capacity of a collagen membrane (3D-COL) enriched with human periodontal-ligament stem cells (hPDLSCs) and CM or EVs or EVs engineered with polyethylenimine (PEI-EVs) in rats subjected to a calvarial defect. We evaluated also their capacity to induce angiogenic factors. At first, in vitro results showed an increased expression of osteogenic markers in hPDLSCs cultured with the 3D-COL and PEI-EVs, associated also with the increased protein levels of Vascular endothelial growth factor (VEGF) and VEGF receptor 2 (VEGFR2). The increased expression of these proteins was confirmed also in vivo in rats implanted with the 3D-COL enriched with hPDLSCs and PEI-EVs. Moreover, histological examination evidenced in this group of rats the activation of bone regeneration and of the vascularization process. Also MicroCT imaging with morphometric analysis confirmed in rats transplanted with 3D-COL enriched with hPDLSCs and PEI-EVs an important regenerative process and a better integration level. All together, these results evidenced that the 3D-COL enriched with hPDLSCs and PEI-EVs may promote bone regeneration of calvaria defects, associated also with an increased vascularization.

Bone tissue regeneration strategies require approaches that provide an osteogenic and angiogenic microenvironment able to drive the bone growth. Recently, the development of 3D printing biomaterials, including poly(lactide) (3D-PLA), enriched with mesenchymal stem cells (MSCs) and/or their derivatives, such as extracellular vesicles (EVs) has been achieving promising results. In this study, in vitro results showed an increased expression of osteogenic and angiogenic markers, as RUNX2, VEGFA, OPN and COL1A1 in the living construct 3D-PLA/human Gingival MSCs (hGMSCs)/EVs. Considering that EVs carry and transfer proteins, mRNA and microRNA into target cells, we evaluated miR-2861 and miR-210 expression related to osteoangiogenesis commitment. Histological examination of rats implanted with 3D-PLA/hGMSCs/EVs evidenced the activation of bone regeneration and of the vascularization process, confirmed also by MicroCT. In synthesis, an upregulation of miR-2861 and -210 other than RUNX2, VEGFA, OPN and COL1A1 was evident in cells cultured in the presence of the biomaterial and EVs. Then, these results evidenced that EVs may enhance bone regeneration in calvaria defects, in association with an enhanced vascularization offering a novel regulatory system in the osteoangiogenesis evolution. The application of new strategies to improve biomaterial engraftment is of great interest in the regenerative medicine and can represent a way to promote bone regeneration.

Bone tissue engineering is based on bone grafting to repair bone defects. Bone graft substitutes can contribute to the addition of mesenchymal stem cells (MSCs) in order to enhance the rate and the quality of defect regeneration. The stem cell secretome contains many growth factors and chemokines, which could affect cellular characteristics and behavior. Conditioned medium (CM) could be used in tissue regeneration avoiding several problems linked to the direct use of MSCs. In this study, we investigated the effect of human periodontal ligament stem cells (hPDLSCs) and their CM on bone regeneration using a commercially available membrane scaffold Evolution (EVO) implanted in rat calvarias. EVO alone or EVO + hPDLSCs with or without CM were implanted in Wistar male rats subjected to calvarial defects. The in vivo results revealed that EVO membrane enriched with hPDLSCs and CM showed a better osteogenic ability to repair the calvarial defect. These results were confirmed by acquired micro-computed tomography (CT) images and the increased osteopontin levels. Moreover, RT-PCR in vitro revealed the upregulation of three genes (Collagen (COL)5A1, COL16A1 and transforming growth factor (TGF)β1) and the down regulation of 26 genes involved in bone regeneration. These results suggest a promising potential application of CM from hPDLSCs and scaffolds for bone defect restoration and in particular for calvarial repair in case of trauma.

Background: The process of osseointegration of dental implants is characterized by healing phenomena at the level of the interface between the surface and the bone. Implant surface modification has been introduced in order to increase the level of osseointegration. The purpose of this study is to evaluate the influence of biofunctional coatings for dental implants and the bone healing response in a rabbit model. The implant surface coated with collagen type I was analyzed through X-ray Photoelectron Spectroscopy (XPS), Atomic Force Microscopy (AFM), micro-CT and histologically. Methods: The sandblasted and double acid etched surface coated with collagen type I, and uncoated sandblasted and double acid etched surface were evaluated by X-ray Photoelectron Spectroscopy (XPS) and Atomic Force Microscopy (AFM) analysis in order evaluate the different morphology. In vivo, a total of 36 implants were positioned in rabbit articular femoral knee-joint, 18 fixtures for each surface. Micro-CT scans, histological and histomorphometrical analysis were conducted at 15, 30 and 60 days. Results: A histological statistical differences were evident at 15, 30 and 60 days (p < 0.001). Both implant surfaces showed a close interaction with newly formed bone. Mature bone appeared in close contact with the surface of the fixture. The AFM outcome showed a similar roughness for both surfaces. Conclusion: However, the final results showed that a coating of collagen type I on the implant surface represents a promising procedure able to improve osseointegration, especially in regions with a low bone quality.

Botulinum neurotoxin type A (BoNT/A) is a major therapeutic agent that has been proven to be a successful treatment for different neurological disorders, with emerging novel therapeutic indications each year. BoNT/A exerts its action by blocking SNARE complex formation and vesicle release through the specific cleavage of SNAP-25 protein; the toxin is able to block the release of pro-inflammatory molecules for months after its administration. Here we demonstrate the extraordinary capacity of BoNT/A to neutralize the complete paralysis and pain insensitivity induced in a murine model of severe spinal cord injury (SCI). We show that the toxin, spinally administered within one hour from spinal trauma, exerts a long-lasting proteolytic action, up to 60 days after its administration, and induces a complete recovery of muscle and motor function. BoNT/A modulates SCI-induced neuroglia hyperreactivity, facilitating axonal restoration, and preventing secondary cells death and damage. Moreover, we demonstrate that BoNT/A affects SCI-induced neuropathic pain after moderate spinal contusion, confirming its anti-nociceptive action in this kind of pain, as well. Our results provide the intriguing and real possibility to identify in BoNT/A a therapeutic tool in counteracting SCI-induced detrimental effects. Because of the well-documented BoNT/A pharmacology, safety, and toxicity, these findings strongly encourage clinical translation.