Xeno-free 3D Culture of Mesenchymal Stromal Cells For Bone Tissue Engineering

Clinical translation of innovative regenerative approaches using mesenchymal stromal cells (MSCs) is urgently needed for the treatment of challenging bone defects. The overall aim of this thesis, comprising of one systematic review and four original studies, was to optimize a xeno-free three-dimensional (3D) culture system of MSCs, as a clinically relevant strategy for bone tissue engineering (BTE). Secondary aims were to identify a minimally invasive source for MSCs, and to promote angiogenesis within the xeno-free 3D cultures. Human platelet lysate (HPL) represents a favourable supplement for xeno-free expansion of MSCs (Study I). To standardize HPL production, the storage time of platelet concentrates was optimized in terms of HPL cytokine content and biological efficacy on MSCs. Advantages of HPL culture (vs. bovine serum) were observed in relation to all relevant in vitro aspects of MSCs, i.e., growth, immunophenotype and osteogenic differentiation (Studies II and III). Progenitor cells showing a characteristic MSC-like phenotype and multipotency were isolated from human gingiva (GPCs) and periodontal ligament (PDLCs). Both GPCs and PDLCs demonstrated superior growth and osteogenic differentiation in HPL vs. FBS; a subset of GPCs also showed potent neurogenic differentiation (Study III). Given their relative ease of isolation and minimally invasive tissue harvesting, GPCs were prioritized in subsequent experiments. To overcome the limitations of traditional monolayer (2D) culture, 3D spheroid cultures were established in HPL. Both GPCs and BMSCs demonstrated significantly increased expression of stemness- and osteogenesis-related genes in spheroids vs. monolayers, confirmed at the protein level by immunocytochemistry. Moreover, the cytokine release profile of GPC and BMSC spheroids was considerably enhanced compared to monolayers. Under osteogenic conditions, GPC spheroids showed in vitro mineralization comparable to that of BMSCs (Study III). When implanted in vivo, xeno-free GPCs and BMSCs showed ectopic mineralization after 4 and 8 weeks based on micro-CT and histology; implanted human cells were identified at the mineralization sites via in situ hybridization. In the case of BMSCs, significantly greater mineralization was observed in constructs containing spheroids vs. single cells (Study V). To enhance angiogenesis, a coculture strategy was tested using a xeno-free spheroid coculture model of GPCs and human umbilical vein ECs (HUVECs) embedded in an HPL-hydrogel (HPLG). When cultured as spheroids, HUVECs showed characteristic in vitro sprouting angiogenesis in HPLG. A trend for increased in vitro HUVEC-sprouting was observed in co-culture with GPCs. Constructs of coculture and HUVEC spheroids in HPLG comparably supported in vivo neoangiogenesis in a chorioallantoic membrane (CAM) assay (Study IV). Clinically relevant BTE constructs were designed combining BMSCs (as spheroids or single cells) encapsulated in HPLG and 3D printed copolymer scaffolds. Viability and osteogenic differentiation of cells within the constructs was confirmed up to 21 days in vitro; greater mineralization was observed in constructs containing spheroids vs. single cells. When implanted in rats’ calvarial defects, constructs of both spheroids and single cells revealed abundant in vivo bone regeneration for up to 12 weeks (Study V). The results herein suggest clear advantages of xeno-free 3D cultures of MSCs for BTE. GPCs represent a promising alternative to BMSCs with osteogenic and proangiogenic potential, and further work is needed to facilitate clinical translation. In particular, the constructs of xeno-free MSCs, HPLG and 3D printed scaffolds developed herein, represent a clinically relevant strategy for BTE

Scaffolds in Periodontal Regenerative Treatment

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Brief communication: Effects of conditioned media from human platelet lysate cultured MSC on osteogenic cell differentiation in vitro.

Culturing mesenchymal stromal cells (MSC) in human platelet lysate (HPL) supplemented media can enhance their osteogenic differentiation potential. The objective of this study was to test the hypothesis that conditioned media (CM) derived from HPL-cultured MSC also have pro-osteogenic effects. Pooled CM was prepared from HPL-cultured human bone marrow MSC (BMSC) of multiple donors and applied on BMSC of different donors (than those used for CM preparation), with or without additional supplementation [HPL, fetal bovine serum (FBS)] and osteogenic stimulation. At various time-points, cell proliferation, alkaline phosphatase (ALP) activity, osteogenic gene expression and in vitro mineralization were assessed. BMSC in standard unstimulated growth media served as controls. After 3-7 days, CM alone did not promote BMSC proliferation or ALP activity; supplementation of CM with HPL slightly improved these effects. After 2 and 7 days, CM alone, but not CM supplemented with HPL, promoted osteogenic gene expression. After 14 days, only CM supplemented with FBS and osteogenic stimulants supported in vitro BMSC mineralization; CM alone and CM supplemented with HPL did not support mineralization, regardless of osteogenic stimulation. In summary, CM from HPL-cultured BMSC promoted osteogenic gene expression but not in vitro mineralization in allogeneic BMSC even when supplemented with HPL and/or osteogenic stimulants. Future studies should investigate the role and relevance of supplementation and osteogenic induction in in vitro assays using CM from MSC

Regeneration of alveolar bone defects in the experimental pig model: A systematic review and meta-analysis.

OBJECTIVE Pigs are emerging as a preferred experimental in vivo model for bone regeneration. The study objective was to answer the focused PEO question: in the pig model (P), what is the capacity of experimental alveolar bone defects (E) for spontaneous regeneration in terms of new bone formation (O)? METHODS Following PRISMA guidelines, electronic databases were searched for studies reporting experimental bone defects or extraction socket healing in the maxillae or mandibles of pigs. The main inclusion criteria were the presence of a control group of untreated defects/sockets and the assessment of regeneration via 3D tomography [radiographic defect fill (RDF)] or 2D histomorphometry [new bone formation (NBF)]. Random effects meta-analyses were performed for the outcomes RDF and NBF. RESULTS Overall, 45 studies were included reporting on alveolar bone defects or extraction sockets, most frequently in the mandibles of minipigs. Based on morphology, defects were broadly classified as 'box-defects' (BD) or 'cylinder-defects' (CD) with a wide range of healing times (10 days to 52 weeks). Meta-analyses revealed pooled estimates (with 95% confidence intervals) of 50% RDF (36.87%-63.15%) and 43.74% NBF (30.47%-57%) in BD, and 44% RDF (16.48%-71.61%) and 39.67% NBF (31.53%-47.81%) in CD, which were similar to estimates of socket-healing [48.74% RDF (40.35%-57.13%) and 38.73% NBF (28.57%-48.89%)]. Heterogeneity in the meta-analysis was high (I2  > 90%). CONCLUSION A substantial body of literature revealed a high capacity for spontaneous regeneration in experimental alveolar bone defects of (mini)pigs, which should be considered in future studies of bone regeneration in this animal model

Proteomic Analysis of Mesenchymal Stromal Cells Secretome in Comparison to Leukocyte- and Platelet-Rich Fibrin.

Secretomes of mesenchymal stromal cells (MSCs) are emerging as a novel growth factor (GF)-based strategy for periodontal and bone regeneration. The objective of this study was to compare the secretome of human bone marrow MSC (BMSC) to that of leukocyte- and platelet-rich fibrin (L-PRF), an established GF-based therapy, in the context of wound healing and regeneration. Conditioned media from human BMSCs (BMSC-CM) and L-PRF (LPRF-CM) were subjected to quantitative proteomic analysis using liquid chromatography with tandem mass spectrometry. Global profiles, gene ontology (GO) categories, differentially expressed proteins (DEPs), and gene set enrichment (GSEA) were identified using bioinformatic methods. Concentrations of selected proteins were determined using a multiplex immunoassay. Among the proteins identified in BMSC-CM (2157 proteins) and LPRF-CM (1420 proteins), 1283 proteins were common. GO analysis revealed similarities between the groups in terms of biological processes (cellular organization, protein metabolism) and molecular functions (cellular/protein-binding). Notably, more DEPs were identified in BMSC-CM (n = 550) compared to LPRF-CM (n = 118); these included several key GF, cytokines, and extracellular matrix (ECM) proteins involved in wound healing. GSEA revealed enrichment of ECM (especially bone ECM)-related processes in BMSC-CM and immune-related processes in LPRF-CM. Similar trends for intergroup differences in protein detection were observed in the multiplex analysis. Thus, the secretome of BMSC is enriched for proteins/processes relevant for periodontal and bone regeneration. The in vivo efficacy of this therapy should be evaluated in future studies

The use of mesenchymal stromal cell secretome to enhance guided bone regeneration in comparison with leukocyte and platelet-rich fibrin.

OBJECTIVES Secretomes of mesenchymal stromal cells (MSC) represent a novel strategy for growth-factor delivery for tissue regeneration. The objective of this study was to compare the efficacy of adjunctive use of conditioned media of bone-marrow MSC (MSC-CM) with collagen barrier membranes vs. adjunctive use of conditioned media of leukocyte- and platelet-rich fibrin (PRF-CM), a current growth-factor therapy, for guided bone regeneration (GBR). METHODS MSC-CM and PRF-CM prepared from healthy human donors were subjected to proteomic analysis using mass spectrometry and multiplex immunoassay. Collagen membranes functionalized with MSC-CM or PRF-CM were applied on critical-size rat calvaria defects and new bone formation was assessed via three-dimensional (3D) micro-CT analysis of total defect volume (2 and 4 weeks) and 2D histomorphometric analysis of central defect regions (4 weeks). RESULTS While both MSC-CM and PRF-CM revealed several bone-related proteins, differentially expressed proteins, especially extracellular matrix components, were increased in MSC-CM. In rat calvaria defects, micro-CT revealed greater total bone coverage in the MSC-CM group after 2 and 4 weeks. Histologically, both groups showed a combination of regular new bone and 'hybrid' new bone, which was formed within the membrane compartment and characterized by incorporation of mineralized collagen fibers. Histomorphometry in central defect sections revealed greater hybrid bone area in the MSC-CM group, while the total new bone area was similar between groups. CONCLUSION Based on the in vitro and in vivo investigations herein, functionalization of membranes with MSC-CM represents a promising strategy to enhance GBR

Bone regeneration in rat calvarial defects using dissociated or spheroid mesenchymal stromal cells in scaffold-hydrogel constructs

Background Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects. The aim of this study was to compare the potential of human BMSC cultured as 2D monolayers or 3D spheroids encapsulated in constructs of 3D-printed poly-L-lactide-co-trimethylene carbonate scaffolds and modified human platelet lysate hydrogels (PLATMC-HPLG) for bone regeneration. Methods PLATMC-HPLG constructs with 2D or 3D BMSC were assessed for osteogenic differentiation based on gene expression and in vitro mineralization. Subsequently, PLATMC-HPLG constructs with 2D or 3D BMSC were implanted in rat calvarial defects for 12 weeks; cell-free constructs served as controls. Bone regeneration was assessed via in vivo computed tomography (CT), ex vivo micro-CT and histology. Results Osteogenic gene expression was significantly enhanced in 3D versus 2D BMSC prior to, but not after, encapsulation in PLATMC-HPLG constructs. A trend for greater in vitro mineralization was observed in constructs with 3D versus 2D BMSC (p > 0.05). In vivo CT revealed comparable bone formation after 4, 8 and 12 weeks in all groups. After 12 weeks, micro-CT revealed substantial regeneration in 2D BMSC (62.47 ± 19.46%), 3D BMSC (51.01 ± 24.43%) and cell-free PLATMC-HPLG constructs (43.20 ± 30.09%) (p > 0.05). A similar trend was observed in the histological analysis. Conclusion Despite a trend for superior in vitro mineralization, constructs with 3D and 2D BMSC performed similarly in vivo. Regardless of monolayer or spheroid cell culture, PLATMC-HPLG constructs represent promising scaffolds for bone tissue engineering applications.publishedVersio

Functionalizing Collagen Membranes with MSC-Conditioned Media Promotes Guided Bone Regeneration in Rat Calvarial Defects.

Functionalizing biomaterials with conditioned media (CM) from mesenchymal stromal cells (MSC) is a promising strategy for enhancing the outcomes of guided bone regeneration (GBR). This study aimed to evaluate the bone regenerative potential of collagen membranes (MEM) functionalized with CM from human bone marrow MSC (MEM-CM) in critical size rat calvarial defects. MEM-CM prepared via soaking (CM-SOAK) or soaking followed by lyophilization (CM-LYO) were applied to critical size rat calvarial defects. Control treatments included native MEM, MEM with rat MSC (CEL) and no treatment. New bone formation was analyzed via micro-CT (2 and 4 weeks) and histology (4 weeks). Greater radiographic new bone formation occurred at 2 weeks in the CM-LYO group vs. all other groups. After 4 weeks, only the CM-LYO group was superior to the untreated control group, whereas the CM-SOAK, CEL and native MEM groups were similar. Histologically, the regenerated tissues showed a combination of regular new bone and hybrid new bone, which formed within the membrane compartment and was characterized by the incorporation of mineralized MEM fibers. Areas of new bone formation and MEM mineralization were greatest in the CM-LYO group. Proteomic analysis of lyophilized CM revealed the enrichment of several proteins and biological processes related to bone formation. In summary, lyophilized MEM-CM enhanced new bone formation in rat calvarial defects, thus representing a novel 'off-the-shelf' strategy for GBR

Genomic analyses of early peri-implant bone healing in humans : a systematic review

ObjectiveThe objective of the study was to systematically review the literature for studies reporting gene expression analyses (GEA) of the biological processes involved in early human peri-implant bone healing.MethodsElectronic databases (MEDLINE, EMBASE) were searched in duplicate. Controlled and uncontrolled studies reporting GEA of human peri-implant tissues - including ≥5 patients and ≥2 time points - during the first 4 weeks of healing were eligible for inclusion. Methodological quality and risk of bias were also assessed.ResultsFour exploratory studies were included in reporting GEA of either tissues attached to SLA or SLActive implants after 4 to 14 days or cells attached to TiOBlast or Osseospeed implants after 3 to 7 days. A total of 111 implants from 43 patients were analyzed using validated array methods; however, considerable heterogeneity and risk of bias were detected. A consistent overall pattern of gene expression was observed; genes representing an immuno-inflammatory response were overexpressed at days 3 to 4, followed by genes representing osteogenic processes at day 7. Genes representing bone remodeling, angiogenesis, and neurogenesis were expressed concomitantly with osteogenesis. Several regulators of these processes, such as cytokines, growth factors, transcription factors, and signaling pathways, were identified. Implant surface properties seemed to influence the healing processes at various stages via differential gene expression.ConclusionLimited evidence from gene expression studies in humans indicates that osteogenic processes commence within the first post-operative week and they appear influenced at various stages by implant surface properties

Volume changes of maxillary sinus augmentations over time : a systematic review

Purpose: The objective of this study was to systematically review the available literature on three-dimensional time-dependent graft volume changes after sinus augmentation (SA) with different biomaterials in humans. Materials and Methods: MEDLINE, EMBASE, and CENTRAL were searched for related literature. Controlled and uncontrolled studies reporting volume changes of more than 10 SAs after at least 6 months, assessed by computed tomography (CT) or cone beam CT (CBCT), were eligible for inclusion. The primary outcome of interest was time-dependent percentage change in augmentation volume. Results: Seven controlled and five uncontrolled studies (n = 234 SAs) with a high risk of bias were included and reported on a range of graft materials. Autogenous bone (AB) was used in the particulate or block form. Bone substitutes (BS) were used either alone or in combination with other materials as composite grafts (CG). All studies reported reductions in augmentation volumes over time (AVR), generally after short observation periods (range, 6 months to 6 years). Substantial AVRs (approximately 45% in 77 SAs) were reported for AB after 6 months and up to 2 years. AVRs for solely BS or CG were relatively lower (approximately 18% to 22% in 142 SAs) after a similar time period. All studies reported a wide range of volume reductions. No significant differences in AVR were observed between different graft materials. Because of insufficient long-term data, a reliable association between volume reduction and time could not be established. Conclusion: Some loss of augmentation volume always occurs after SA during early healing times. In general, less AVR may be expected after SA with BS or CG compared to SA with AB. Augmentation volume loss does not seem to compromise implant placement or survival