Extracellular Vesicle-Based Hydrogels for Wound Healing Applications.

Hydrogels and extracellular vesicle-based therapies have been proposed as emerging therapeutic assets in wound closure. The combination of these elements has given good results in managing chronic and acute wounds. The intrinsic characteristics of the hydrogels in which the extracellular vesicles (EVs) are loaded allow for overcoming barriers, such as the sustained and controlled release of EVs and the maintenance of the pH for their conservation. In addition, EVs can be obtained from different sources and through several isolation methods. However, some barriers must be overcome to transfer this type of therapy to the clinic, for example, the production of hydrogels containing functional EVs and identifying long-term storage conditions for EVs. The aim of this review is to describe the reported EV-based hydrogel combinations, along with the obtained results, and analyze future perspectives.

Evaluation of Platelet-Derived Extracellular Vesicles in Gingival Fibroblasts and Keratinocytes for Periodontal Applications.

Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates such as PL. In this article, the use of extracellular vesicles (EVs) derived from platelet lysate (PL) and their combination with hyaluronic acid biomaterials (HA) in an in vitro wound healing assay is investigated. EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro in gingival fibroblasts and keratinocytes for biocompatibility (LDH activity and metabolic activity) and by an in vitro wound-healing assay and gene expression analysis. EVs and EVs-HA treatments were biocompatible in gingival fibroblasts and keratinocytes and showed an increase in wound healing in vitro compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed after the treatment with EVs. EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet-derived EVs could emerge as a new application for periodontal regeneration in combination with biomaterials in order to enhance their clinical use.

Platelet-Derived Extracellular Vesicles for Regenerative Medicine.

Extracellular vesicles (EVs) present a great potential for the development of new treatments in the biomedical field. To be used as therapeutics, many different sources have been used for EVs obtention, while only a few studies have addressed the use of platelet-derived EVs (pEVs). In fact, pEVs have been shown to intervene in different healing responses, thus some studies have evaluated their regenerative capability in wound healing or hemorrhagic shock. Even more, pEVs have proven to induce cellular differentiation, enhancing musculoskeletal or neural regeneration. However, the obtention and characterization of pEVs is widely heterogeneous and differs from the recommendations of the International Society for Extracellular Vesicles. Therefore, in this review, we aim to present the main advances in the therapeutical use of pEVs in the regenerative medicine field while highlighting the isolation and characterization steps followed. The main goal of this review is to portray the studies performed in order to enhance the translation of the pEVs research into feasible therapeutical applications.

Enhanced osteoinductive capacity and decreased variability by enrichment of demineralized bone matrix with a bone protein extract.

Osteoinductive capacity of demineralized bone matrix (DBM) is sometimes insufficient or shows high variability between different batches of DBM. Here, we tried to improve its osteoinductive activity by alkali-urea or trypsin treatment but this strategy was unsuccessful. Then, we tested the enrichment of DBM with a bone protein extract (BPE) containing osteogenic growth factors comparing two sources: cortical bone powder and DBM. The osteoinductive capacity (alkaline phosphatase activity) of the obtained BPEs was evaluated in vitro in C2C12 cells. Specific protein levels present in the different BPE was determined by enzyme-linked immunosorbent assay or by a multiplex assay. BPE from cortical bone powder showed a lack of osteoinductive effect, in agreement with the low content on osteoinductive factors. In contrast, BPE from DBM showed osteoinductive activity but also high variability among donors. Thus, we decided to enrich DBM with BPE obtained from a pool of DBM from different donors. Following this strategy, we achieved increased osteoinductive activity and lower variability among donors. In conclusion, the use of a BPE obtained from a pool of demineralized bone to enrich DBM could be used to increase its osteoinductive effect and normalize the differences between donors.

Quercitrin Nanocoated Implant Surfaces Reduce Osteoclast Activity In Vitro and In Vivo.

In this study, the effect on osteoclast activity in vitro and in vivo of titanium implants that were coated with quercitrin was evaluated. Titanium surfaces were covalently coated with the flavonoid quercitrin. The effect of the surfaces on osteoclastogenesis was first tested in vitro on RAW264.7 cells that were supplemented with receptor activator of nuclear factor kappa-B ligand (RANKL) to generate osteoclast-like cells by tartrate-resistant acid phosphatase (TRAP) inmunostaining after five days of culture, and by analysis of the mRNA expression levels of markers related to bone resorption after seven days of culture. A rabbit tibial model was used to evaluate the in vivo biological response to the implant surfaces after eight weeks of healing, analyzing the lactate dehydrogenase (LDH) and the alkaline phosphatase (ALP) activities in the wound fluid that were present at the implant interface and the peri-implant bone mRNA expression levels of several markers related to inflammation, bone resorption and osteoblast-osteoclast interaction. No differences between groups and control surfaces were found in the wound fluid analyses. Moreover, quercitrin implant surfaces significantly decreased the expression of osteoclast related genes in vitro (Trap, CalcR, Ctsk, H⁺ATPase, Mmp9) and in vivo (Ctsk, H⁺ATPase, Mmp9) as well as the expression of RankL in vivo. Moreover, quercitrin surfaces were not cytotoxic for the cells. Thus, quercitrin implant surfaces were biocompatible and decreased osteoclastogenesis in vitro and in vivo. This could be used to improve the performance of dental implants.

Titanium implants coated with UV-irradiated vitamin D precursor and vitamin E: in vivo performance and coating stability.

OBJECTIVES: This study aimed at evaluating the biological response of titanium implants coated with UV-irradiated 7-dehydrocholesterol (7-DHC) and vitamin E (VitE) in vivo and analyzing the effects of aging on their stability and bioactivity in vitro. MATERIAL AND METHODS: Titanium surfaces were coated with 7-DHC and VitE, UV-irradiated and incubated for 48 h at 23°C to allow cholecalciferol synthesis. The in vivo biological response was tested using a rabbit tibia model after 8 weeks of healing by analyzing the wound fluid and the mRNA levels of several markers at the bone-implant interface (N = 8). The stability of the coating after storage up to 12 weeks was determined using HPLC analysis, and the bioactivity of the stored modified implants was studied by an in vitro study with MC3T3-E1 cells (N = 6). RESULTS: A significant increase in gene expression levels of osteocalcin was found in the bone tissue attached to implants coated with the low dose of 7-DHC and VitE, together with a higher ALP activity in the wound fluid. Implants treated with the high dose of 7-DHC and VitE showed increased tissue necrosis and inflammation. Regarding the aging effects, coated implants were stable and bioactive up to 12 weeks when stored at 4°C and avoiding oxygen, light and moisture. CONCLUSION: This study demonstrates that Ti implants coated with UV-irradiated 7-DHC and VitE promote in vivo gene expression of bone formation markers and ALP activity, while they keep their osteopromotive potential in vitro and composition when stored up to 12 weeks at 4°C.

Gene expression and morphometric parameters of human bone biopsies after maxillary sinus floor elevation with autologous bone combined with Bio-Oss® or BoneCeramic®.

OBJECTIVES: Although the clinical success of Bio-Oss(®) and BoneCeramic(®) has been corroborated by histologic and histomorphometric findings, the biological events that occur during healing after maxillary sinus floor elevation (MSFE) are unknown. Here, we evaluated biopsies of grafted bone with a mixture of autologous bone and Bio-Oss(®) or BoneCeramic(®) after two different healing time periods to understand the molecular process underlying bone formation after MSFE. MATERIAL AND METHODS: Seven patients, following a bilateral split-mouth design model and needing a MSFE to allow implant placement, were recruited for this study. Right or left sinuses were grafted with autologous maxillary bone combined either with Bio-Oss(®) or BoneCeramic(®) , respectively. Twenty biopsies were taken at the time of implant insertion after 4-5 months or 6-8 months of MSFE, and analyzed by micro-computed tomography (microCT) and gene-expression analysis. RESULTS: MicroCT analysis revealed no differences in the morphometric parameters or BMD either after 4-5 months or 6-8 months of MSFE between Bio-Oss(®) and BoneCeramic(®) . At molecular level, a higher expression of bone forming gene Runx2 was observed after 4-5 months of MSFE in the Bio-Oss(®) compared with the BoneCeramic(®) group. CONCLUSIONS: Our results indicate that differences found at the molecular level between Bio-Oss(®) and BoneCeramic(®) are not translated to important differences in the 3D microstructure and BMD of the grafted bone.

Nanostructured Implant-Tissue Interface Assessment Using a Three-Dimensional Gingival Tissue Equivalent.

Improved soft tissue integration (STI) around dental implants is key for implant success. The formation of an early and long-lasting transmucosal seal around the implant abutment might help to prevent peri-implantitis, one of the major causes of late implant failure. In natural teeth, collagen fibers are firmly inserted and fixed in the cementum of the tooth and emerge perpendicular to the gingival tissue. In contrast, around dental implants, collagen fibers run predominantly parallel to the implant surface, allowing bacterial migration into the peri-implant interface that might lead to peri-implantitis. Previous studies have shown that nanostructured Ti surfaces improve gingival cell response in monolayer cell cultures. Here, we aimed at evaluating the implant-tissue interface using a 3D gingival tissue equivalent (GTE). First, we evaluated the GTE response to a nanostructured (NN) and machined Ti surface after the stimulation with Porphyromonas gingivalis lipopolysaccharide (LPS), to simulate peri-implantitis conditions. Thus, GTE viability, through MTT assay, the release of metalloproteinase-1 (MMP1) and its inhibitor (TIMP1) through ELISA, and the gene expression of extracellular matrix turnover genes by real-time RT-PCR were analyzed. Second, GTE-implant interaction was characterized by serial block face scanning electron microscopy, and collagen-1 orientation at the tissue-implant interface was analyzed by immunofluorescence. While a similar GTE response to LPS stimulation was found for both implant surfaces, a higher proportion of collagen oriented perpendicular to the implant was observed on the NN implant surface. Thus, our results indicate that the nanostructuration of titanium dental implant abutments could allow the correct orientation of collagen fibers and greater soft tissue sealing, while keeping biocompatibility levels and LPS response comparable.

UV-irradiated 7-dehydrocholesterol coating on polystyrene surfaces is converted to active vitamin D by osteoblastic MC3T3-E1 cells.

The aim of the present study was to determine the effects of UV irradiation on the conversion of 7-dehydrocholesterol (7-DHC), which has been coated onto a polystyrene surface, to cholecalciferol (D3), and the resulting effect on the formation of vitamin D (1,25-D3) by MC3T3-E1 cells. The changes in gene expression of the enzymes regulating its hydroxylation, Cyp27b1 and Cyp27a1, were monitored as well as the net effect of the UV-treated 7-DHC coating on cell viability and osteoblast differentiation. MC3T3-E1 cells were found to express the enzymes required for synthesizing active 1,25-D3, and we found a dose-dependent increase in the production of both 25-D3 and 1,25-D3 levels for UV-activated 7-DHC samples unlike UV-untreated ones. Cell viability revealed no cytotoxic effect for any of the treatments, but only for the highest dose of 7-DHC (20 nmol per well) that was UV-irradiated. Furthermore, osteoblast differentiation was increased in cells treated with some of the higher doses of 7-DHC when UV-irradiated, as shown by collagen-I, osterix and osteocalcin relative mRNA levels. The conversion of 7-DHC to preD3 exogenously by UV irradiation and later to 25-D3 by MC3T3-E1 cells was determined for the optimum 7-DHC dose (0.2 nmol per well), i.e. 8.6 ± 0.7% of UV-activated 7-DHC was converted to preD3 and 6.7 ± 2.8% of preD3 was finally converted to 25-D3 under the conditions studied. In conclusion, we demonstrate that an exogenous coating of 7-DHC, when UV-irradiated, can be used to endogenously produce active vitamin D. We hereby provide the scientific basis for UV-activated 7-DHC coating as a feasible approach for implant therapeutics focused on bone regeneration.

Correlation between molecular signals and bone bonding to titanium implants.

OBJECTIVES: A better understanding of the biological processes controlling osseointegration at the bone-to-implant interface is needed. The aim of this study was to examine which are the molecular and biochemical variables that are significantly related to osseointegration, using multiple regression analysis. MATERIALS AND METHODS: Titanium coins were placed into the tibial cortical bone of New Zealand White rabbits and evaluated using pull-out test after 4 and 8 weeks of healing. Correlations between pull-out and several markers from tissue fluid (Lactate dehydrogenase [LDH] and Alkaline phosphatase [ALP] activities and total protein content) and peri-implant bone tissue (total protein, RNA and DNA content, implant area covered with bone and gene expression of osteoblast, osteoclast and inflammation markers) were used to assess the importance of these parameters in bone healing and in relation to implant performance. RESULTS: Our results showed a negative correlation between the content of DNA, RNA and total protein at the peri-implant bone tissue and the pull-out force, indicating that as bone matures and implant becomes more osseointegrated, the organic content of bone decreases. The negative correlation found between pull-out force and ALP activity pointed to a delayed healing in implants with lower pull-out values and primary mineralization still ongoing. LDH activity and total protein content in the tissue fluid were as well negatively correlated with the pull-out force. Finally, a positive correlation was observed between the pull-out force and the expression of the osteoblast and the bone resorption markers, being osteocalcin and collagen-I the best predictive markers for osseointegration after 4 and 8 weeks of healing respectively. CONCLUSIONS: These results suggest that the evaluation of these markers could be relevant for the assessment of new implant surfaces for rapid bone healing and improved implant performance.

Comparative effect of platelet- and mesenchymal stromal cell-derived extracellular vesicles on human cartilage explants using an ex vivo inflammatory osteoarthritis model.

Aims: Extracellular vesicles (EVs) are nanoparticles secreted by all cells, enriched in proteins, lipids, and nucleic acids related to cell-to-cell communication and vital components of cell-based therapies. Mesenchymal stromal cell (MSC)-derived EVs have been studied as an alternative for osteoarthritis (OA) treatment. However, their clinical translation is hindered by industrial and regulatory challenges. In contrast, platelet-derived EVs might reach clinics faster since platelet concentrates, such as platelet lysates (PL), are already used in therapeutics. Hence, we aimed to test the therapeutic potential of PL-derived extracellular vesicles (pEVs) as a new treatment for OA, which is a degenerative joint disease of articular cartilage and does not have any curative or regenerative treatment, by comparing its effects to those of human umbilical cord MSC-derived EVs (cEVs) on an ex vivo OA-induced model using human cartilage explants. Methods: pEVs and cEVs were isolated by size exclusion chromatography (SEC) and physically characterized by nanoparticle tracking analysis (NTA), protein content, and purity. OA conditions were induced in human cartilage explants (10 ng/ml oncostatin M and 2 ng/ml tumour necrosis factor alpha (TNFα)) and treated with 1 × 109 particles of pEVs or cEVs for 14 days. Then, DNA, glycosaminoglycans (GAG), and collagen content were quantified, and a histological study was performed. EV uptake was monitored using PKH26 labelled EVs. Results: Significantly higher content of DNA and collagen was observed for the pEV-treated group compared to control and cEV groups. No differences were found in GAG quantification nor in EVs uptake within any treated group. Conclusion: In conclusion, pEVs showed better performance than cEVs in our in vitro OA model. Although further studies are needed, pEVs are shown as a potential alternative to cEVs for cell-free regenerative medicine.

Platelet-derived extracellular vesicles formulated with hyaluronic acid gels for application at the bone-implant interface: An animal study.

Background/Objective: Platelet derived extracellular vesicles (pEV) are promising therapeutical tools for bone healing applications. In fact, several in vitro studies have already demonstrated the efficacy of Extracellular Vesicles (EV) in promoting bone regeneration and repair in various orthopedic models. Therefore, to evaluate the translational potential in this field, an in vivo study was performed. Methods: Here, we used hyaluronic acid (HA) gels formulated with pEVs, as a way to directly apply pEVs and retain them at the bone defect. In this study, pEVs were isolated from Platelet Lysate (PL) through size exclusion chromatography and used to formulate 2% HA gels. Then, the gels were locally applied on the tibia cortical bone defect of New Zeland White rabbits before the surgical implantation of coin-shaped titanium implants. After eight weeks, the bone healing process was analyzed through biomechanical, micro-CT, histological and biochemical analysis. Results: Although no biomechanical differences were observed between pEV formulated gels and non-formulated gels, biochemical markers of the wound fluid at the interface presented a decrease in Lactate dehydrogenase (LDH) activity and alkaline phosphatase (ALP) activity for pEV HA treated implants. Moreover, histological analyses showed that none of the treatments induced an irritative effect and, a decrease in the fibrotic response surrounding the implant for pEV HA treated implants was described. Conclusion: In conclusion, pEVs improve titanium implants biocompatibility at the bone-implant interface, decreasing the necrotic effects of the surgery and diminishing the fibrotic layer associated to the implant encapsulation that can lead to implant failure.

Differential response of MC3T3-E1 and human mesenchymal stem cells to inositol hexakisphosphate.

BACKGROUND: Inositol hexakisphosphate (IP6) has been found to have an important role in biomineralization. METHODS: Because the complete mechanism of action of IP6 on osteoblasts is not fully understood and its potential use in the primary prevention of osteoporosis, we examined the direct effect of IP6 on cell viability and differentiation of MC3T3-E1 cells and on differentiation of human umbilical cord mesenchymal stem cells (hUC-MSCs). RESULTS: We show that IP6 has different effects depending on the origin of the cell target. Thus, while IP6 decreased gene expression of osteoblast markers and mineralization in MC3T3-E1 cells without negatively affecting cell viability and ALP activity, an increase in gene expression of ALP was observed in hUC-MSCs committed to the osteoblastic lineage. This increasing effect of IP6 on ALP mRNA expression levels was reversed by the addition of a selective inhibitor of IP6 kinase, suggesting that the effect of IP6 might be due through its pyrophosphorylated derivatives. Besides, Rankl mRNA levels were decreased after IP6 treatment in MC3T3-E1 cells, pointing to a paracrine effect on osteoclasts. CONCLUSION: Our results indicate that IP6 has different effects on osteoblast differentiation depending on the cell type and origin. However, further studies are needed to examine the net effect of IP6 on bone formation and its potential as novel antiosteoporosis drug.

Smicl is required for phosphorylation of RNA polymerase II and affects 3'-end processing of RNA at the midblastula transition in Xenopus.

Smicl (Smad-interacting CPSF 30-like) is an unusual protein that interacts with transcription factors as well as with the cleavage and polyadenylation specificity factor (CPSF). Previous work has shown that Smicl is expressed maternally in the Xenopus embryo and is later required for transcription of Chordin. In this paper we search for additional targets of Smicl. We identify many genes whose onset of expression at the midblastula transition (MBT) requires Smicl and is correlated with the translocation of Smicl from cytoplasm to nucleus. At least one such gene, Xiro1, is regulated via 3'-end processing. In searching for a general mechanism by which Smicl might regulate gene expression at the MBT, we have discovered that it interacts with the tail of Rpb1, the largest subunit of RNA polymerase II. Our results show that Smicl is required for the phosphorylation of the Rpb1 tail at serine 2 of the repeated heptapeptide YSPTSPS. This site becomes hyperphosphorylated at the MBT, thus allowing the docking of proteins required for elongation of transcription and RNA processing. Our work links the onset of zygotic gene expression in the Xenopus embryo with the translocation of Smicl from cytoplasm to nucleus, the phosphorylation of Rpb1 and the 3'-end processing of newly transcribed mRNAs.

Sinus graft with safescraper: 5-year results.

PURPOSE: In the procedure of sinus floor elevation, autogenous bone, allogenic grafts, and several other bone substitutes are used. However, autogenous bone is still considered the gold standard. Donor sites for autogenous bone are generally the iliac crest, oral cavity, calvarium bone, and tibia. In this work the experience with the use of a Safescraper device for harvesting of autogenous bone is reported and a decision-making algorithm for grafting in sinus floor elevation procedures is proposed. MATERIALS AND METHODS: Forty sinus augmentation procedures were performed in 34 patients. All sinuses were filled with a mixture of autogenous bone and bovine hydroxyapatite. A Safescraper device was used to harvest autologous bone from the maxillary area. Platelet-rich plasma was used to sustain bone placement. Sixty-five dental implants were placed at 4 months with a flapless procedure. A clinical and radiological 5-year retrospective case series of a cohort is reported. RESULTS: In all cases new bone formation was confirmed radiologically and implant placement was performed successfully. Analysis of samples obtained by biopsy with histology and microcomputed tomography showed the presence of mature bone. Healing problems were observed in only 1 case. CONCLUSIONS: Sinus augmentation with bone grafts obtained from oral cavity with a bone scraper device has the advantage of providing autogenous bone without the need for an extra surgical approach. This procedure yields satisfactory results in bone formation, implant survival, and patient satisfaction. When combined with a flapless approach for implant placement, a decrease in the morbidity of the entire process is achieved.

Multifunctional Properties of Quercitrin-Coated Porous Ti-6Al-4V Implants for Orthopaedic Applications Assessed In Vitro.

(1) One strategy to improve the outcome of orthopedic implants is to use porous implants with the addition of a coating with an antibacterial biomolecule. In this study, we aimed to produce and test the biocompatibility, the osteopromotive (both under normal conditions and under a bacterial challenge with lipopolysaccharide (LPS)) and antibacterial activities of a porous Ti-6Al-4V implant coated with the flavonoid quercitrin in vitro. (2) Porous Ti-6Al-4V implants were produced by 3D printing and further functionalized with quercitrin by wet chemistry. Implants were characterized in terms of porosity and mechanical testing, and the coating with quercitrin by fluorescence staining. Implant biocompatibility and bioactivity was tested using MC3T3-E1 preosteoblasts by analyzing cytotoxicity, cell adhesion, osteocalcin production, and alkaline phosphatase (ALP) activity under control and under bacterial challenging conditions using lipopolysaccharide (LPS). Finally, the antibacterial properties of the implants were studied using Staphylococcus epidermidis by measuring bacterial viability and adhesion. (3) Porous implants showed pore size of about 500 µm and a porosity of 52%. The coating was homogeneous over all the 3D surface and did not alter the mechanical properties of the Young modulus. Quercitrin-coated implants showed higher biocompatibility, cell adhesion, and osteocalcin production compared with control implants. Moreover, higher ALP activity was observed for the quercitrin group under both normal and bacterial challenging conditions. Finally, S. epidermidis live/dead ratio and adhesion after 4 h of incubation was lower on quercitrin implants compared with the control. (4) Quercitrin-functionalized porous Ti-6Al-4V implants present a great potential as an orthopedic porous implant that decreases bacterial adhesion and viability while promoting bone cell growth and differentiation.

Improved physical and osteoinductive properties of demineralized bone matrix by gelatin methacryloyl formulation.

The demineralized bone matrix (DBM) is the most widely used bone allograft, which is obtained by removing the mineral component of bone, leading to exposure of the proteins responsible for osteoinduction. For clinical use, DBM shall be formulated with a carrier that provides consistency and improves its osteoinduction. In this study, three DBM formulations with glycerol (Gly), hyaluronic acid (HA), and gelatin methacryloyl (GelMA) were evaluated measuring their physicochemical properties (microstructure, compressive strength, and serum cohesivity) and their osteoinductive capacity both in vitro using C2C12 cells and umbilical cord human mesenchymal stem cells and in vivo in an ectopic bone formation model in athymic mice. To assess the effectiveness of DBM in vitro in inducing the differentiation into osteoblasts, alkaline phosphatase (ALP) activity was assessed in combination with a cytotoxicity assay. In vivo, new bone formation was assessed by histological and radiological analysis. In the compression and in the serum cohesive assays, the GelMA DBM formulation showed its superiority over the other formulations. In addition, GelMA showed a more compact structure analysed by scanning electron microscopy. Higher cell toxicity was observed on Gly formulations in vitro, whereas GelMa and HA showed very low toxicity. All formulations significantly improved ALP activity compared with control. In the in vivo studies, GelMA showed the greatest osteoinductive potential with a higher percentage of new bone and bone marrow formation. Our results suggest GelMA is useful as a carrier for DBM designed to promote the formation of the new bone.

Platelet-derived extracellular vesicles promote osteoinduction of mesenchymal stromal cells.

AIMS: Platelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs). METHODS: EVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and the expression of CD9 and CD63 markers by western blot. The effect of the obtained EVs on osteoinduction was evaluated in vitro on human umbilical cord MSCs by messenger RNA (mRNA) expression analysis of bone markers, alkaline phosphatase activity (ALP), and calcium (Ca2+) content. RESULTS: Osteogenic differentiation of MSCs was confirmed when treated with UC-isolated EVs. In order to disprove that the effect was due to co-isolated proteins, EVs were isolated by SEC. Purer EVs were obtained and proved to maintain the differentiation effect on MSCs and showed a dose-dependent response. CONCLUSION: PL-derived EVs present an osteogenic capability comparable to PL treatments, emerging as an alternative able to overcome PL and PRP limitations.Cite this article: Bone Joint Res 2020;9(10):667-674.

Biomimetic Biomolecules in Next Generation Xeno-Hybrid Bone Graft Material Show Enhanced In Vitro Bone Cells Response.

Bone defects resulting from trauma, disease, surgery or congenital malformations are a significant health problem worldwide. Consequently, bone is the second most transplanted tissue just after blood. Although bone grafts (BGs) have been used for decades to improve bone repairs, none of the currently available BGs possesses all the desirable characteristics. One way to overcome such limitations is to introduce the feature of controlled release of active bone-promoting biomolecules: however, the administration of, e.g., recombinant Bone morphogenetic proteins (BMPs) have been used in concentrations overshooting physiologically occurring concentrations and has thus raised concerns as documented side effects were recorded. Secondly, most such biomolecules are very sensitive to organic solvents and this hinders their use. Here, we present a novel xeno-hybrid bone graft, SmartBonePep®, with a new type of biomolecule (i.e., intrinsically disordered proteins, IDPs) that is both resistant to processing with organic solvent and both triggers bone cells proliferation and differentiation. SmartBonePep® is an advanced and improved modification of SmartBone®, which is a bone substitute produced by combining naturally-derived mineral bone structures with resorbable polymers and collagen fragments. Not only have we demonstrated that Intrinsically Disordered Proteins (IDPs) can be successfully and safely loaded onto a SmartBonePep®, withstanding the hefty manufacturing processes, but also made them bioavailable in a tuneable manner and proved that these biomolecules are a robust and resilient biomolecule family, being a better candidate with respect to other biomolecules for effectively producing the next generation bone grafts. Most other biomolecules which enhances bone formation, e.g., BMP, would not have tolerated the organic solvent used to produce SmartBonePep®.