Sarah Al-Maawi

In vivo cellular reactions to different biomaterials—Physiological and pathological aspects and their consequences

Biomaterials are widely used in guided bone regeneration (GBR) and guided tissue regeneration (GTR). After application, there is an interaction between the host immune system and the implanted biomaterial, leading to a biomaterial-specific cellular reaction. The present review focuses on cellular reactions to numerous biomaterials in vivo with consideration of different implantation models and microenvironments in different species, such as subcutaneous implantation in mice and rats, a muscle model in goats and a femur model in rabbits. Additionally, cellular reactions to different biomaterials in various clinical indications within the oro-maxillofacial surgical field were considered. Two types of cellular reactions were observed. There was a physiological reaction with the induction of only mononuclear cells and a pathological reaction with the induction of multinucleated giant cells (MNGCs). Attention was directed to the frequently observed MNGCs and consequences of their appearance within the implantation region. MNGCs have different subtypes. Therefore, the present review addresses the different morphological phenotypes observed within the biomaterial implantation bed and discusses the critical role of MNGCs, their subtypes and their precursors as well as comparing the characteristics and differences between biomaterial-related MNGCs and osteoclasts. Polymeric biomaterials that only induced mononuclear cells underwent integration and maintained their integrity, while polymeric biomaterials that induced MNGCs underwent disintegration with material breakdown and loss of integrity. Hence, there is a question regarding whether our attention should be directed to alternative biological concepts, in combination with biomaterials that induce a physiological mononuclear cellular reaction to optimize biomaterial-based tissue regeneration.

Reduction of the relative centrifugal force influences cell number and growth factor release within injectable PRF-based matrices

Platelet rich fibrin (PRF) is a blood concentrate system obtained by centrifugation of peripheral blood. First PRF matrices exhibited solid fibrin scaffold, more recently liquid PRF-based matrix was developed by reducing the relative centrifugation force and time. The aim of this study was to systematically evaluate the influence of RCF (relative centrifugal force) on cell types and growth factor release within injectable PRF- in the range of 60–966 g using consistent centrifugation time. Numbers of cells was analyzed using automated cell counting (platelets, leukocytes, neutrophils, lymphocytes and monocytes) and histomorphometrically (CD 61, CD- 45, CD-15+, CD-68+, CD-3+ and CD-20). ELISA was utilized to quantify the concentration of growth factors and cytokines including PDGF-BB, TGF-β1, EGF, VEGF and MMP-9. Leukocytes, neutrophils, monocytes and lymphocytes had significantly higher total cell numbers using lower RCF. Whereas, platelets in the low and medium RCF ranges both demonstrated significantly higher values when compared to the high RCF group. Histomorphometrical analysis showed a significantly high number of CD61+, CD-45+ and CD-15+ cells in the low RCF group whereas CD-68+, CD-3+ and CD-20+ demonstrated no statistically significant differences between all groups. Total growth factor release of PDGF-BB, TGF-β1 and EGF had similar values using low and medium RCF, which were both significantly higher than those in the high RCF group. VEGF and MMP-9 were significantly higher in the low RCF group compared to high RCF. These findings support the LSCC (low speed centrifugation concept), which confirms that improved PRF-based matrices may be generated through RCF reduction. The enhanced regenerative potential of PRF-based matrices makes them a potential source to serve as a natural drug delivery system. However, further pre-clinical and clinical studies are required to evaluate the regeneration capacity of this system.Graphical abstract

A low-speed centrifugation concept leads to cell accumulation and vascularization of solid platelet-rich fibrin: an experimental study in vivo

Abstract Platelet-rich fibrin (PRF) is generated from the patients’ own venous blood by a single centrifugation step without the additional use of anticoagulants. Based on the previously described LSCC (low-speed centrifugation concept), our group showed that modification of the centrifugation setting, that is, reducing the relative centrifugal force (RCF) and mildly increasing the centrifugation time, resulted in modified solid and liquid PRF-matrices with increased number of platelets, leukocytes, and growth factors’ concentrations. The aim of this study was to determine whether RCF reduction might also result in different tissue reactions toward the two PRF-based matrices, especially vascularization and cell distribution in vivo. Two centrifugation protocols (PRF-high [719 g] and PRF-medium [222 g]) were compared in a subcutaneous implantation model of SCID mice at 5 and 10 days. Histological and histomorphometrical analyses were performed to quantify lymphocyte, neutrophil, human macrophage, and monocyte populations. CD31 was used to detect newly formed vessels, while all human cells were detected by using human vimentin as a pan-cellular marker. The results demonstrated that PRF-high elicited a dense and stable fibrin structure and prevented cellular penetration of the host tissue. By contrast, PRF-medium was more porous, had a significantly higher in vivo vascularization rate, and included significantly more human cells, especially at day 10, compared to PRF-high. These findings highlight the possibility of modifying the structure and composition of PRF matrices and thus selectively altering their regenerative potential in vivo. Clinical studies now must evaluate the different PRF matrices for bone and soft-tissue regeneration to validate possible benefits using personalized preparation protocols.

Influence of concentration and preparation of platelet rich fibrin on human bone marrow mononuclear cells (in vitro)

Abstract Large bone defects have always been a big challenge. The use of bone marrow mononuclear cells (BMCs) combined with an osteoconductive scaffold has been proved a good alternative for the treatment of large bone defects. Another autologous source for tissue engineering is platelet rich fibrin (PRF). PRF is a blood concentrate system obtained through a one-step centrifugation. The generated 3D matrix of the PRF clot serves as a reservoir of growth factors. Those growth factors might support the regenerative response of BMC, and therefore the effect of PRF, centrifuged with either high medium (208 g) or low (60 g) relative centrifugation force (RCF) on BMCs was evaluated in vitro in the present study. The two PRF matrices obtained were initially characterized and compared to human serum. Significantly increased concentrations of insulin-like growth factor (IGF), soluble intercellular adhesion molecule-1 (sICAM1) and transforming growth factor (TGF)-β were found in PRF compared to human serum whereas VEGF concentration was not significantly altered. A dose-response study revealed no further activation of BMC’s metabolic activity, if concentration of both PRF matrices exceeded 10% (v/v). Effect of both PRF preparations [10%] on BMC was analyzed after 2, 7, and 14 days in comparison to human serum [10%]. Metabolic activity of BMC increased significantly in all groups on day 14. Furthermore, gene expression of matrix metalloproteinases (MMP)-2, −7, and −9 was significantly stimulated in BMC cultivated with the respective PRF matrices compared to human serum. Apoptotic activity of BMC incubated with PRF was not altered compared to BMC cultivated with serum. In conclusion, PRF could be used as a growth factor delivery system of autologous or allogeneic source with the capability of stimulating cells such as BMC.

Individualized titanium mesh combined with platelet-rich fibrin and deproteinized bovine bone: A new approach for challenging augmentation

Autologous bone transfer is regarded as the gold standard for ridge augmentation before dental implantation, especially in severe bony defects caused by tumor resection or atrophy. In addition to the advantages of autologous bone, transplantation has several disadvantages, such as secondary operation, increased morbidity and pain. The present study reports, for the first time, a combination of a xenogeneic bone substitute (BO) with platelet-rich fibrin (PRF), which is a fully autologous blood concentrate derived from the patients own peripheral blood by centrifugation. Solid A-PRF+ and liquid i-PRF together with an individualized 3-D planned titanium mesh were used for reconstruction of a severe tumor-related bony defect within the mandible of a former head and neck cancer patient. The BO enriched with regenerative components from PRF allowed the reconstruction of the mandibular resective defect under the 3-D mesh without autologous bone transplantation. Complete rehabilitation and restoration of the patients oral function were achieved. Histological analysis of extracted bone biopsies confirmed that the new bone within the augmented region originated from the residual bone. Within the limitations of the presented case, the applied concept appears to be a promising approach to increase the regenerative capacity of a bone substitute material, as well as decrease the demand for autologous bone transplantation, even in cases in which autologous bone is considered the golden standard. PRF can be considered a reliable source for increasing the biological capacities of bone substitute materials.

Homogeneous pressure influences the growth factor release profiles in solid platelet-rich fibrin matrices and enhances vascular endothelial growth factor release in the solid platelet-rich fibrin plugs

Aims: Platelet-rich fibrin (PRF) exists in both solid and fluid forms. The present study was the first to evaluate the influence of homogeneous pressure on the growth factor (GF) release in pressed PRF-matrices and plugs. Methods and Material: A solid PRF-matrix (208 g; 8 min) was pressed to obtain a plug, and a pressed PRF-matrix that are used in clinical application. The released exudates were evaluated compared to liquid PRF (60 g and 3 min). The VEGF, TGF-ß1 and EGF release was quantified using ELISA. The fibrin structure and cellular components in solid PRF groups were evaluated histologically. Results: The pressed PRF-matrix and PRF-plug exhibited denser fibrin structure compared to the non-pressed PRF-matrix. On day 7, the PRF-plug and non-pressed PRF-matrix showed significantly higher release of VEGF, TGF-ß1 and EGF compared to that of the pressed PRF-matrix. The accumulated VEGF concentration was significantly higher in the PRF-plug compared to that in the PRF-matrix and non-pressed PRF-matrix. The accumulated EGF and TGF-ß1 concentrations over 10 days showed no statistically significant differences between the evaluated solid PRF groups. The exudates released TGF-ß1 and EGF passively, that was only detectable in after 1 and 7 hours. Liquid PRF released significantly higher GFs than the exudates at all investigated time points. The early VEGF and EGF release in liquid PRF (1 hour to 1 day) was significantly higher than that in the solid PRF-matrices. On day 10, significantly higher accumulated GFs were detected in the solid PRF groups compared to those in the liquid PRF. Thus, the combination of both solid and liquid PRF is a potential tool to generate a clinically relevant system with sustained bioactivity. Conclusions: These results highlight the potential to influence the GFs release profile of solid PRF matrices by pressure and obtain a clinically applicable plug with significantly higher VEGF release, providing further understanding of the release profile of PRF matrices as a drug delivery system.

In vivo Implantation of a Bovine-Derived Collagen Membrane Leads to Changes in the Physiological Cellular Pattern of Wound Healing by the Induction of Multinucleated Giant Cells: An Adverse Reaction?

The present study evaluated the tissue response toward a resorbable collagen membrane derived from bovine achilles tendon (test group) in comparison to physiological wound healing (control group). After subcutaneous implantation in Wistar rats over 30 days, histochemical and immunohistochemical methods elucidated the cellular inflammatory response, vascularization pattern, membrane protein and cell absorbance capacity. After 30 days, the test-group induced two different inflammatory patterns. On the membrane surface, multinucleated giant cells (MNGCs) were formed after the accumulation of CD-68-positive cells (macrophages), whereas only mononuclear cells (MNCs) were found within the membrane central region. Peri-implant vascularization was significantly enhanced after the formation of MNGCs. No vessels were found within the central region of the membrane. Physiological wound healing revealed no MNGCs at any time point. These dynamic changes in the cellular reaction and vascularization within the test-group are related typical indications of a foreign body reaction. Due to the membrane-specific porosity, mononuclear cells migrated into the central region, and the membrane maintained its integrity over 30 days by showing no breakdown or disintegration. The ex vivo investigation analyzed the interaction between the membrane and a blood concentrate system, liquid platelet-rich fibrin (liquid PRF), derived from human peripheral blood and consisting of platelets, leukocytes and fibrin. PRF penetrated the membrane after just 15 min. The data question the role of biomaterial-induced MNGCs as a pathological reaction and whether this is acceptable to trigger vascularization or should be considered as an adverse reaction. Therefore, further pre-clinical and clinical studies are needed to identify the types of MNGCs that are induced by clinically approved biomaterials.

Variant Purification of an Allogeneic Bone Block

Objective This short communication reports on a histological analysis of the composition of the commercially available Maxgraft® allogeneic bone block. Materials and Methods Based on previously published, easily applicable histological methods, blanc samples of the Maxgraft® allogeneic bone block have been decalcified, dehydrated and embedded in paraffin before histological and histochemical staining. Afterwards, the slides were evaluated for their material characteristics, such as the bone matrix structure and other components, including collagen or cells/cell remnants. Results The results show that this bone block exhibits a trabecular structure with lamellar sub-organization. Additionally, cellular remnants within the osteocyte lacunae and at the outer trabecular surfaces reside together with remnants of the former inter-trabecular fatty and connective tissue, i.e., collagenous structures and connective tissue cells or cell remnants. Conclusion Consistent with a previous study on this topic, the data presented here demonstrate that some of the certified purification techniques might not allow for the production of allogeneic materials free of organic cell and tissue components.