Sebastian Blatt

Purpose: Biocompatible collagen membranes (CM) are widely used in regenerative dentistry, particularly in guided tissue regeneration (GTR) and guided bone regeneration (GBR). While resorbable CMs offer advantages such as reduced patient morbidity and enhanced wound healing, their barrier function can impede vascularization, potentially compromising graft survival. Biofunctionalization of CMs with platelet-rich fibrin (PRF) and hyaluronic acid (HA) has shown promise in enhancing angiogenesis. This experimental study evaluates the pro-angiogenic effects of biofunctionalizing collagen membranes with advanced PRF (A-PRF), injectable PRF (i-PRF), and HA using the chorioallantoic membrane (CAM) assay in ovo. Methods: Three porcine-derived collagen membranes (Mucoderm®, Bio-Gide®, and Smartbrane®) were biofunctionalized with A-PRF, i-PRF, or HA and applied to the CAM assay on day 7 of incubation. Afterwards, two experimental series were evaluated. The first series investigated Mucoderm (MM native, A-PRF, and MM combined with A-PRF) over an observation period ranging from 24 to 120 h ( N = 135). The second series examined Bio-Gide® and Smartbrane (BM native, BM combined with i-PRF, BM combined with HA, and SM combined with HA) over a period from 24 to 72 h ( N = 60). To assess impact on angiogenesis, vascularization was evaluated at multiple time points (24 h, 48 h, 72 h, 96 h, and 120 h) using immunohistochemical staining (hematoxylin-eosin, α-smooth muscle actin, CD105) and artificial intelligence (AI)-assisted image analysis (IKOSA® software). Results: Biofunctionalization of Mucoderm® with A-PRF significantly enhanced angiogenesis up to 96 h, as evidenced by increased vessel area, length, and branching points ( p < 0.05). I-PRF biofunctionalization of Bio-Gide® also promoted angiogenesis between 24 h ( p = 0.036) and 72 h, showing significantly improved values for total area ( p = 0.007), vessel length ( p = 0.018), and vessel thickness ( p = 0.008) compared with the native membrane. While HA biofunctionalization of Bio-Gide® and Smartbrane® resulted in significantly increased angiogenesis at 48 and 72 h ( p < 0.05), its effects were less pronounced than those achieved with PRF variants. Native Bio-Gide® exhibited greater pro-angiogenic potential than native Mucoderm® at 24 h ( p = 0.012); however, biofunctionalized membranes generally outperformed native variants. Conclusions: Biofunctionalization of collagen membranes with A-PRF and i-PRF significantly enhances angiogenesis in ovo, with A-PRF showing sustained effects up to 96 h. HA also promotes angiogenesis and represents a viable, cost-effective alternative that does not require blood collection. Both PRF and HA biofunctionalization may offer potential benefits for enhancing vascularization in GBR/GTR applications. However, their pro-angiogenic potential and clinical relevance remain exploratory at this stage for pourely in-ovo results. The study is limited to an in-ovo setting, therefore the transferability to oral soft tissue and bone conditions is restricted. Further randomized clinical studies are needed to better understand and evaluate the possible advantages.

Background: The computer‐aided design/computer‐aided manufacturing (CAD/CAM) workflow is state of the art for a plethora of oral‐ and maxillofacial reconstruction procedures. The workflow is well implemented in alveolar ridge preservation and segmental bone regeneration. Guided bone or guided tissue regeneration (GBR/GTR) are essential in stabilising and regenerating the alveolar ridge height and width and the surrounding soft tissue in segmental deficient situations. CAD/CAM‐assisted GBR minimises surgical time, optimises bone volume preservation and provides predictable results in implantology and reconstructive procedures. Besides, the technique is crucial in planning and executing free microvascular anastomosed tissue transfer for continuity defects. Methods: This review examines how CAD/CAM fabrication can further transform bony regeneration and reconstruction. Therefore, latest principles of CAD/CAM fabrication methods, manufacturing techniques in CAD/CAM bone fabrication, materials for CAD/CAM‐fabricated bone in GBR, clinical applications of CAD/CAM bone in GBR, challenges and limitations as well as future perspectives are highlighted. Results and Conclusions: In conclusion, CAD/CAM is poised to become a standard approach not only for segmental but also continuity defect situations in regenerative maxillofacial surgery as technology evolves.

Differences in cell count and growth factor expression between first- and second-generation autologous platelet concentrates (APCs) have been well described. The debate over which formula best supports wound healing in various surgical procedures is still ongoing. This study aims to assess the whole proteome assembly, cell content, immunological potential and pro-angiogenic potential of second-generation APC, Platelet-Rich Fibrin (PRF) vs. first-generation APC, Platelet-Rich Plasma (PRP). The global proteome of the APCs was analyzed using nano-liquid chromatography mass spectrometry. Blood cell concentrations were determined by an automated cell counter. The effect of APCs on macrophage polarization was analyzed by flow cytometry. A yolk sac membrane (YSM) assay was used to monitor the neo-vessel formation and capillary branching in vivo. Cell count analysis revealed a higher number/concentration of leukocytes in PRF vs. PRP. Incubation of macrophages with PRP or platelet-free plasma (PFP) did not induce a significant pro-inflammatory state but led to a shift to the M0/M2 phenotype as seen in wound healing for all tested formulas. Label-free proteomics analysis identified a total of 387 proteins from three biological replicates of the respective designated groups. PRF induced increased formation of neo-vessels and branching points in vivo in comparison to PRP and PFP (each p < 0.001), indicating the enhanced pro-angiogenic potential of PRF. Overall, PRF seems superior to PRP, an important representative of first-generation formulas. Inclusion of leucocytes in PRF compared to PRP suggested rather an anti-inflammatory effect on macrophages. These results are important to support the versatile clinical applications in regenerative medicine for second-generation autologous platelet concentrates to optimize wound healing.

Introduction: Autologous platelet concentrate (APC) are pro-angiogenic and can promote wound healing and tissue repair, also in combination with other biomaterials. However, challenging defect situations remain demanding. 3D bioprinting of an APC based bioink encapsulated in a hydrogel could overcome this limitation with enhanced physio-mechanical interface, growth factor retention/secretion and defect-personalized shape to ultimately enhance regeneration.Methods: This study used extrusion-based bioprinting to create a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate. Chemico-physical testing exhibited an amorphous structure characterized by high shape fidelity. Cytotoxicity assay and incubation of human osteogenic sarcoma cells (SaOs2) exposed excellent biocompatibility. enzyme-linked immunosorbent assay analysis confirmed pro-angiogenic growth factor release of the printed constructs, and co-incubation with HUVECS displayed proper cell viability and proliferation. Chorioallantoic membrane (CAM) assay explored the pro-angiogenic potential of the prints in vivo. Detailed proteome and secretome analysis revealed a substantial amount and homologous presence of pro-angiogenic proteins in the 3D construct.Results: This study demonstrated a 3D bioprinting approach to fabricate a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate with high shape fidelity, biocompatibility, and substantial pro-angiogenic properties.Conclusion: This approach may be suitable for challenging physiological and anatomical defect situations when translated into clinical use.

Objectives: Pre-vascularization of the collagen membranes with autologous platelet concentrates is a standard procedure in oral and maxillofacial surgery. This study analyzed the possible interaction of an acellular collagen membrane of porcine origin (NM) with platelet-rich fibrin (PRF) regarding its rehydration protocol with differences in pH values and effect on angiogenesis. Materials and methods: NM was analyzed alone and combined with solid PRF by plotting or co-culturing with injectable PRF. Different media (venous blood, buffer solution with a fixed pH value of 7, saline solution, and injectable PRF) were used to analyze the influence on pH value during rehydration. Chorion allantois membrane assay (CAM) was applied to check pro-angiogenic effects after 24, 48, and 72 h, followed by immunohistochemical analysis. Results: Rehydration in injectable PRF showed acidity over time (p < 0.05). A definite pro-angiogenic effect of NM alone was found regarding neo-vessel formation supported by the respective light microscopically analysis without significant differences to PRF alone (p > 0.005). This pro-angiogenic effect could not be exaggerated when NM was combined with liquid/solid PRF (each p > 0.005). Conclusions: Rehydration with liquid PRF of the collagen membrane results in acidity compared to a saline solution or patient’s blood. The significant pro-angiogenic potential of the membrane alone resulted in enhanced neo-vessel formation that could not be optimized with the addition of PRF. Clinical relevance statement: Using injectable PRF for rehydration protocol of the collagen membrane leads to acidosis that can ultimately optimize wound healing. Differences in the physio-mechanical interplay of collagen matrices and autologous platelet concentrates must result in clinical algorithms if pre-vascularization can maximize outcomes.

Free flap tissue transfer represents the gold standard for extensive defect reconstruction, although malperfusion due to thrombosis remains the leading risk factor for flap failure. Recent studies indicate an increased immune response and platelet activation in connection with pathologic coagulation. The underlying cellular and molecular mechanisms remain poorly understood, however. The presented study, therefore, aims to investigate if transfer-related ischemia alters intra-flap metabolism and electrolyte concentrations compared to central venous blood after free flap transfer in pigs to establish a novel experimental model. Free transfer of a myocutaneous gracilis flap to the axillary region was conducted in five juvenile male pigs. The flap artery was anastomosed to the axillary artery, and intra-flap venous blood was drained and transfused using a rubber-elastic fixed intravenous catheter. Blood gas analysis was performed to assess the effect of transfer time-induced ischemia on intra-flap electrolyte levels, acid–base balance, and hemoglobin concentrations compared to central venous blood. Time to flap reperfusion was 52 ± 10 min on average, resulting in a continuous pH drop (acidosis) in the flaps’ venous blood compared to the central venous system (p = 0.037). Potassium (p = 0.016), sodium (p = 0.003), and chloride (p = 0.007) concentrations were significantly increased, whereas bicarbonate (p = 0.016) and calcium (p = 0.008) significantly decreased within the flap. These observations demonstrate the induction of anaerobic glycolysis and electrolyte displacement resulting in acidosis and hence significant tissue damage already after a short ischemic period, thereby validating the novel animal model for investigating intra-flap metabolism and offering opportunities for exploring various (immuno-) thrombo-hemostatic issues in transplantation surgery.

Improved serological biomarkers are needed for the early detection, risk stratification and treatment surveillance of patients with oral squamous cell carcinoma (OSCC). We performed an exploratory study using advanced, highly specific, DNA-aptamer-based serum proteomics (SOMAscan, 1305-plex) to identify distinct proteomic changes in patients with OSCC pre- vs. post-resection and compared to healthy controls. A total of 63 significantly differentially expressed serum proteins (each p < 0.05) were found that could discriminate between OSCC and healthy controls with 100% accuracy. Furthermore, 121 proteins were detected that were significantly altered between pre- and post-resection sera, and 12 OSCC-associated proteins reversed to levels equivalent to healthy controls after resection. Of these, 6 were increased and 6 were decreased relative to healthy controls, highlighting the potential relevance of these proteins as OSCC tumor markers. Pathway analyses revealed potential pathophysiological mechanisms associated with OSCC. Hence, quantitative proteome analysis using SOMAscan technology is promising and may aid in the development of defined serum marker assays to predict tumor occurrence, progression and recurrence in OSCC, and to guide personalized therapies.