Upscaling Osteoclast Generation by Enhancing Macrophage Aggregation Using Hollow Microgels.

Osteoclasts, the bone resorbing cells of hematopoietic origin formed by macrophage fusion, are essential in bone health and disease. However, in vitro research on osteoclasts remains challenging due to heterogeneous cultures that only contain a few multinucleated osteoclasts. Indeed, a strategy to generate homogeneous populations of multinucleated osteoclasts in a scalable manner has remained elusive. Here, the investigation focuses on whether microencapsulation of human macrophages in microfluidically generated hollow, sacrificial tyramine-conjugated dextran (Dex-TA) microgels could facilitate macrophage precursor aggregation and formation of multinucleated osteoclasts. Therefore, human mononuclear cells are isolated from buffy coats and differentiated toward macrophages. Macrophages are encapsulated in microgels using flow focus microfluidics and outside-in enzymatic oxidative phenolic crosslinking, and differentiated toward osteoclasts. Morphology, viability, and osteoclast fusion of microencapsulated cells are assessed. Furthermore, microgels are degraded to allow cell sorting of released cells based on osteoclastic marker expression. The successful encapsulation and osteoclast formation of human macrophages in Dex-TA microgels are reported for the first time using high-throughput droplet microfluidics. Intriguingly, osteoclast formation within these 3D microenvironments occurs at a significantly higher level compared to the conventional 2D culture system. Furthermore, the feasibility of establishing a pure osteoclast culture from cell transfer and release from degradable microgels is demonstrated.

The effect of surface roughening on the success of orthodontic mini-implants: A systematic review and meta-analysis.

INTRODUCTION: Orthodontic mini-implants are a widely accepted treatment modality in orthodontics; however, the failure rate is moderately high. Surface roughening is the golden standard in conventional oral implantology, and this may prove beneficial for orthodontic mini-implants as well. The objective of this systematic review is to assess the effect of surface roughening on the success rate of orthodontic mini-implants in both adolescent and adult patients undergoing orthodontic treatment. METHODS: Randomized studies comparing the success of surface-roughened and smooth, machined-surface orthodontic mini-implants were included. A literature search was conducted for 6 electronic databases (Pubmed/Medline, Embase, Cochrane, CINAHL, Web of Science, and Scopus), Clinical trial registry (https://www. CLINICALTRIALS: gov), and grey literature (Google Scholar). A manual search of the reference lists of included studies was performed. Two authors independently performed the screening, data extraction, risk of bias, and quality assessments. The risk of bias was assessed with the Cochrane risk-of-bias 2.0 Tool. Data were synthesized using a random effect model meta-analysis presented as a forest plot. The certainty in the body of evidence was assessed using the Grading of Recommendations Assessment, Development, and Evaluation tool. RESULTS: A total of 4226 unique records were screened, and 6 of these were included in the quantitative analysis. Four additional articles were selected for a secondary outcome. A total of 364 orthodontic mini-implants were included in the primary outcome analysis. There was no statistically significant effect of surface roughening on the success of orthodontic mini-implants (odds ratio = 0.63 favoring roughened orthodontic mini-implants; 95% confidence interval, 0.35-1.14). The secondary outcome (ie, the overall failure rate of roughened orthodontic mini-implants) was 6% based on studies with high heterogeneity. Limitations of this study were the risk of bias, study imprecision, and possible publication bias, leading to a very low certainty in the body of evidence. CONCLUSIONS: There is very low-quality evidence that there is no statistically significant effect of surface roughening on the success of orthodontic mini-implants in humans. The overall failure rate of surface-roughened orthodontic mini-implants was 6%. FUNDING: No funding was received for this review. REGISTRATION: This study was preregistered in the Prospective Register of Systematic Reviews (CRD42022371830).

Biomaterials-aided mandibular reconstruction using in vivo bioreactors.

Large mandibular defects are clinically challenging to reconstruct due to the complex anatomy of the jaw and the limited availability of appropriate tissue for repair. We envision leveraging current advances in fabrication and biomaterials to create implantable devices that generate bone within the patients themselves suitable for their own specific anatomical pathology. The in vivo bioreactor strategy facilitates the generation of large autologous vascularized bony tissue of customized geometry without the addition of exogenous growth factors or cells. To translate this technology, we investigated its success in reconstructing a mandibular defect of physiologically relevant size in sheep. We fabricated and implanted 3D-printed in vivo bioreactors against rib periosteum and utilized biomaterial-based space maintenance to preserve the native anatomical mandibular structure in the defect site before reconstruction. Nine weeks after bioreactor implantation, the ovine mandibles were repaired with the autologous bony tissue generated from the in vivo bioreactors. We evaluated tissues generated in bioreactors by radiographic, histological, mechanical, and biomolecular assays and repaired mandibles by radiographic and histological assays. Biomaterial-aided mandibular reconstruction was successful in a large superior marginal defect in five of six (83%) sheep. Given that these studies utilized clinically available biomaterials, such as bone cement and ceramic particles, this strategy is designed for rapid human translation to improve outcomes in patients with large mandibular defects.

Biomaterial-based possibilities for managing peri-implantitis.

Peri-implantitis is an inflammatory disease of hard and soft tissues around osseointegrated implants, followed by a progressive damage of alveolar bone. Oral microorganisms can adhere to all types of surfaces by the production of multiple adhesive factors. Inherent properties of materials will influence not only the number of microorganisms, but also their profile and adhesion force onto the material surface. In this perspective, strategies to reduce the adhesion of pathogenic microorganisms on dental implants and their components should be investigated in modern rehabilitation concepts in implant dentistry. To date, several metallic nanoparticle films have been developed to reduce the growth of pathogenic bacteria. However, the main drawback in these approaches is the potential toxicity and accumulative effect of the metals over time. In view of biological issues and in attempt to prevent and/or treat peri-implantitis, biomaterials as carriers of antimicrobial substances have attracted special attention for application as coatings on dental implant devices. This review will focus on biomaterial-based possibilities to prevent and/or treat peri-implantitis by describing concepts and dental implant components suitable for engagement in preventing and treating this disease. Additionally, we raise important criteria referring to the geometric parameters of dental implants and their components, which can directly affect peri-implant tissue conditions. Finally, we overview currently available biomaterial systems that can be used in the field of oral implantology.

Periodontal regeneration via chemoattractive constructs.

AIM: Chemoattractants, such as stromal cell-derived factor-1α (SDF-1α), can offer an advantage for periodontal regeneration by recruiting the patient's own stem cells to stimulate self-repair. We here developed a chemoattractive construct for periodontal regeneration using SDF-1α and evaluated its efficacy in vivo. MATERIALS AND METHODS: SDF-1α was loaded on gelatin sponge and tested in vitro for SDF-1α release. Subsequently, SDF-1α constructs were implanted into rat periodontal defects for 1 and 6 weeks, with unloaded materials and empty defects as controls. The regenerative efficacy was evaluated by micro-CT, histological and histomorphometrical analyses. RESULTS: In vitro results showed limited SDF-1α release up to 35 days. In contrast, SDF-1α constructs significantly improved periodontal defect regeneration in terms of alveolar bone height, new bone area and functional ligament length. Additionally, SDF-1α constructs decreased the inflammatory response at Week 6. CONCLUSION: Chemoattractive constructs significantly improved periodontal regeneration in terms of alveolar bone height, new bone area and functional ligament length.

Macrophage type modulates osteogenic differentiation of adipose tissue MSCs.

Since the reconstruction of large bone defects remains a challenge, knowledge about the biology of bone healing is desirable to develop novel strategies for improving the treatment of bone defects. In osteoimmunology, macrophages are the central component in the early stage of physiological response after bone injury and bone remodeling in the late stage. During this process, a switch of macrophage phenotype from pro-inflammatory (M1) to anti-inflammatory (M2) is observed. An appealing option for bone regeneration would be to exploit this regulatory role for the benefit of osteogenic differentiation of osteoprogenitor cells (e.g., mesenchymal stem cells; MSCs) and to eventually utilize this knowledge to improve the therapeutic outcome of bone regenerative treatment. In view of this, we focused on the in vitro interaction of different macrophage subtypes with adipose tissue MSCs to monitor the behavior (i.e. proliferation, differentiation and mineralization) of the latter in dedicated co-culture models. Our data show that co-culture of MSCs with M2 macrophages, but not with M1 macrophages or M0 macrophages, results in significantly increased MSC mineralization caused by soluble factors. Specifically, M2 macrophages promoted the proliferation and osteogenic differentiation of MSCs, while M0 and M1 macrophages solely stimulated the osteogenic differentiation of MSCs in the early and middle stages during co-culture. Secretion of the soluble factors oncostatin M (OSM) and bone morphogenetic protein 2 (BMP-2) by macrophages showed correlation with MSC gene expression levels for OSM-receptor and BMP-2, suggesting the involvement of both signaling pathways in the osteogenic differentiation of MSCs.

Comparison of different surface modifications for titanium implants installed into the goat iliac crest.

OBJECTIVES: This in vivo study with implants installed in the goat iliac crest was performed to determine whether the biological and mechanical properties of the bone-to-implant interface are influenced by (i) the type of implant anchorage (i.e., mono- vs. bicortical placement), and (ii) the presence of a bioactive hydroxyapatite (HA) or composite HA/bioactive glass (BG) coatings. MATERIALS AND METHODS: A total of 96 titanium (Ti) implants w/- coatings (Ti, Ti-HA & Ti-HABG; n = 8) were mono- or bicortically placed in the iliac crest of eight goats. At installation and after 4 weeks, implant stability was determined using insertion and removal torque testing (ITQ & RTQ). The peri-implant bone response was histologically and histomorphometrically evaluated by means of bone-to-implant contact (BIC%) and bone area (BA%). RESULTS: Monocortical implants demonstrated significantly lower RTQ values in comparison to ITQ values, whereas for bicortical implant placement RTQ and ITQ were similar. Further, mean RTQ values for monocortical implants were significantly lower in comparison to bicortical implants. Histomorphometrical evaluation demonstrated higher BIC% and BA% for bicortical implants compared to monocortical implants. For bicortical implants, BA% in the inner peri-implant region (0-500 µm) was significantly higher compared to the middle (500-1000 µm) and outer (1000-1500 µm) region. Also, a significant correlation was observed for monocortical implants between RTQ and BIC% and BA%. For surface modifications, no significant differences were found in ITQ and RTQ, for neither mono- nor bicortical implants. Histomorphometrically, HABG-coated implants demonstrated significantly higher BIC% compared to GAE surfaces for both mono- and bicortical implants. Bicortical HA-coated implants revealed significant higher BA% in the inner peri-implant region (0-500 µm) in comparison to bicortical GAE implants. CONCLUSIONS: This study demonstrated that bicortical implant placement beneficially affects implant stability during the early phase of osseointegration. A significant correlation between removal torque and bone-to-implant contact and bone area for monocortical implants was observed, but not for bicortical implants. Therefore, histomorphometrical data should be interpreted with caution to predict the biomechanical implant fixation of bone implants over time. Regarding surface modifications, in the present implantation model, the addition of BG to an RF magnetron sputtered HA coating enhanced the biological behavior of the coating compared to grit-blasted/acid-etched implants.

Comparative evaluation of the combined application of titanium implants and calcium phosphate bone substitutes in a rabbit model.

OBJECTIVES: To study the healing of defects around titanium implants filled with biphasic calcium phosphates (BCP). MATERIAL AND METHODS: Forty custom-made, titanium implants (Ti) with a diameter of 5 mm, and length of 8 mm, with two-sided gaps, were fabricated and installed in the femoral condyle of 20 rabbits. Following a randomization protocol, implants were alternately installed in one condyle without BCP bone substitute material (Ti) in the gaps and in the contralateral condyle gaps were filled with BCP bone substitute material (Ti+BCP). The implants were retrieved after 4 and 12 weeks of healing, after which histological and histomorphometrical analyses were done to assess the percentage of bone implant contact (BIC), the percentage of bone area (BA) and the percentage of particle area (PA) within the region of interest (ROI); the rectangular area joining the two arms of the L-shaped implant was considered as the ROI. RESULTS: After 4 and 12 weeks of healing, Ti+BCP showed significantly higher BIC and BA values compared to Ti. Further, the BCP particles showed a significant decrease from 4 to 12 weeks of healing. The BCP particles (PA) showed a significant reduction from 31.6 ± 11.0% at 4 weeks to 21.0 ± 7.2% at 12 weeks. CONCLUSION: The addition of BCP bone substitute to fill peri-implant gaps significantly enhanced both bone formation (~2.5-fold) and bone to implant contact (>2-fold) for the custom-made titanium implants with two-sided gaps.

In vivo evaluation of bioactive glass-based coatings on dental implants in a dog implantation model.

OBJECTIVES: Although titanium is commonly used as a favorable bone implant material due to its mechanical properties, its bioactive and osteoconductive capacity is relatively low. Calcium phosphate ceramics, predominantly hydroxyapatite (HA), have been frequently used for coating purposes to improve the bioactive properties. In view of the suggested osteopromotive capacity of bioactive glasses (BGs), this study aimed to evaluate the effect of BG incorporation into HA coatings on implant performance in terms of bone contact and bone area. MATERIALS AND METHODS: A total of 48 screw-type titanium implants with magnetron sputter coatings containing different ratios of HA and BG (HA, HABGLow, and HABGHigh; n = 8) were placed into the mandible of 16 Beagle dogs. After 4 and 12 weeks, their performance was evaluated histologically and histomorphometrically. Peri-implant bone area percentage (BA%) was determined in three zones (inner, 0-500 µm; middle, 500-1000 µm; and outer, 1000-1500 µm). Additionally, bone-to-implant contact (BIC%) and first bone-implant contact (1st BIC) were assessed for each sample. RESULTS: After 4 weeks, bone-to-implant contact for the HA- and HABGLow-coated groups was significantly higher (P < 0.05) than for the HABGHigh coatings. Mean values for overall BA% showed comparable values for both the HABGLow (58.3%)- and HABGHigh (56.3%)-coated groups. Data suggest that the relative BA around the HA-coated implants (67.8%) was higher, although this was only significant compared to the HABGHigh group. After 12 weeks, all three groups showed similar bone-to-implant contact and no differences in BA were found. CONCLUSIONS: The incorporation of BG into HA sputter coatings did not enhance the performance of a dental implant in implantations sites with good bone quality and quantity. On the contrary, coatings containing high concentrations of BG resulted in inferior performance during the early postimplantation healing phase.

Modelling bone metastasis in spheroids to study cancer progression and screen cisplatin efficacy.

Most bone metastases are caused by primary breast or prostate cancer cells settling in the bone microenvironment, affecting normal bone physiology and function and reducing 5-year survival rates to 10% and 6%, respectively. To expedite clinical availability of novel and effective bone metastases treatments, reliable and predictive in vitro models are urgently required to screen for novel therapies as current in vitro 2D planar mono-culture models do not accurately predict the clinical efficacy. We herein engineered a novel human in vitro 3D co-culture model based on spheroids to study dynamic cellular quantities of (breast or prostate) cancer cells and human bone marrow stromal cells and screen chemotherapeutic efficacy and specificity of the common anticancer drug cisplatin. Bone metastatic spheroids (BMSs) were formed rapidly within 24 h, while the morphology of breast versus prostate cancer BMS differed in terms of size and circularity upon prolonged culture periods. Prestaining cell types prior to BMS formation enabled confocal imaging and quantitative image analysis of in-spheroid cellular dynamics for up to 7 days of BMS culture. We found that cancer cells in BMS proliferated faster and were less susceptible to cisplatin treatment compared to 2D control cultures. Based on these findings and the versatility of our methodology, BMS represent a feasible 3D in vitro model for screening of new bone cancer metastases therapies.

Osmolarity-Induced Altered Intracellular Molecular Crowding Drives Osteoarthritis Pathology.

Osteoarthritis (OA) is a multifactorial degenerative joint disease of which the underlying mechanisms are yet to be fully understood. At the molecular level, multiple factors including altered signaling pathways, epigenetics, metabolic imbalance, extracellular matrix degradation, production of matrix metalloproteinases, and inflammatory cytokines, are known to play a detrimental role in OA. However, these factors do not initiate OA, but are mediators or consequences of the disease, while many other factors causing the etiology of OA are still unknown. Here, it is revealed that microenvironmental osmolarity can induce and reverse osteoarthritis-related behavior of chondrocytes via altered intracellular molecular crowding, which represents a previously unknown mechanism underlying OA pathophysiology. Decreased intracellular crowding is associated with increased sensitivity to proinflammatory triggers and decreased responsiveness to anabolic stimuli. OA-induced lowered intracellular molecular crowding could be renormalized via exposure to higher extracellular osmolarity such as those found in healthy joints, which reverse OA chondrocyte's sensitivity to catabolic stimuli as well as its glycolytic metabolism.

Maxillary sinus floor augmentation with injectable calcium phosphate cements: a pre-clinical study in sheep.

OBJECTIVES: The aim of this pre-clinical study was to evaluate the biological performance of two injectable calcium phosphate cement (CPC) composite materials containing poly(D,L-lactic-co-glycolic)acid (PLGA) microspheres with different properties in a maxillary sinus floor elevation model in sheep. MATERIALS AND METHODS: PLGA microspheres were made of either low molecular weight (~17 kDa) acid-terminated PLGA (PLGA(L-AT) ) or high molecular weight (~44 kDa) end-capped PLGA (PLGA(H-EC) ) and incorporated in CPC. Eight female Swifter sheep underwent a bilateral maxillary sinus floor elevation procedure via an extra-oral approach. All animals received both materials, alternately injected in the left and right sinus (split-mouth model) and a time point of 12 weeks was used. Analysis of biological performance was based on histology, histomorphometry, and evaluation of sequential fluorochrome labeling. RESULTS: Both types of CPC-PLGA composites showed biocompatibility and direct bone-cement contact. CPC-PLGA(L-AT) showed a significantly higher degradation distance compared to CPC-PLGA(H-EC) (1949 ± 1295 µm vs. 459 ± 267 µm; P = 0.0107). Further, CPC-PLGA(L-AT) showed significantly more bone in the region of interest (26.4 ± 10.5% vs. 8.6 ± 3.9% for PLGA(H-EC) ; P = 0.0009) and significantly less remaining CPC material (61.2 ± 17.7% vs. 81.9 ± 10.9% for PLGA(H-EC) ; P = 0.0192). CONCLUSIONS: Both CPC-PLGA(L-AT) and CPC-PLGA(H-EC) demonstrated to be safe materials for sinus floor elevation procedures in a large animal model, presenting biocompatibility and direct bone contact. In view of material performance, CPC-PLGA(L-AT) showed significantly faster degradation and a significantly higher amount of newly formed bone compared to CPC-PLGA(H-EC) .

Long-term survival of calcium phosphate-coated dental implants: a meta-analytical approach to the clinical literature.

BACKGROUND: Calcium phosphate ceramic coatings have the potential to compensate for challenging bone conditions such as delayed or impaired bone healing and low bone quantity or density. Thus, the increasing universal prevalence of subjects with such challenging bone conditions might be paralleled by an enhanced global use of calcium phosphate ceramic-coated dental implants. However, it is speculated that the long-term clinical survival of calcium phosphate-coated dental implants might be adversely affected by coating delamination. OBJECTIVE: The aims of the current review were (1) to systematically appraise and (2) to meta-analyse long-term survival data of calcium phosphate-coated dental implants in clinical trials. MATERIALS AND METHODS: An extensive search in the electronic databases of the National Library of Medicine (http://www.ncbi.nlm.nih.gov), The Cochrane Central Register of Controlled Trials and the ISI Web of Knowledge, was carried out for articles published between January 2000 and November 2011 to identify randomized controlled clinical trials, prospective clinical trials as well as retrospective analysis of cases (RA) presenting survival data on the topic of calcium phosphate-coated dental implants. Only publications in English were considered, and the search was narrowed to studies in humans with a follow-up of at least 5 years only. Furthermore, the reference lists of related review articles and publications selected for inclusion in this review were systematically screened. The primary outcome variable was percentage annual failure rate (AFR), and the secondary outcome variable was percentage cumulative survival rate (CSR). RESULTS: The electronic search in the database of the National Library of Medicine, The Cochrane Central Register of Controlled Trials and the ISI Web of Knowledge, resulted in the identification of 385 titles. These titles were initially screened by the two independent reviewers for possible inclusion, resulting in 29 publications suitable for further consideration. Screening the abstracts led to 20 full-text articles. From these articles, 15 reports were excluded. Finally, five of these original research reports could be selected for evaluation. No additional publications were identified by manual search. Thus, a total of five articles were included for analysis. Meta-analysis revealed that neither AFRs of calcium phosphate-coated dental implants increased progressively nor that long-term CSRs for calcium phosphate-coated dental implants were inferior to survival rates of noncoated implants. CONCLUSION: We conclude that (1) published long-term survival data for calcium phosphate-coated dental implants are very limited, (2) AFRs of calcium phosphate-coated dental implants do not increase progressively, and (3) long-term CSRs for calcium phosphate-coated dental implants are comparable to survival rates of noncoated implants.

Biological response to titanium implants coated with nanocrystals calcium phosphate or type 1 collagen in a dog model.

OBJECTIVE: The current study aimed to evaluate the osteogenic potential of electrosprayed organic and non-organic surface coatings in a gap-implant model over 4 and 12 weeks of implantation into the dog mandible. MATERIAL AND METHODS: Sixteen Beagle dogs received experimental titanium implants in the mandible 3 months after removal of left premolars (P2, P3 and P4). Three types of implants were installed in each animal: non-coated implant, nano-CaP coated implant and implant with type 1 collagen coating. Both micro-CT and histomorphometry were used to evaluate peri-implant bone response after implantation periods of 4 and 12 weeks. The bone area percentage was assessed histomorphometrically in three different zones (inner: 0-300 µm; middle: 300-600 µm; and outer: 600-1000 µm) around the implant surface. Bone-bridging of the gap was also calculated for each sample. RESULTS: Four weeks after implantation, nano-CaP and collagen-coated implants showed significantly higher bone volume (BV) in the inner zone compared with non-coated implants (P < 0.05 and P < 0.01). After 12 weeks, histomorphometric analysis showed comparable amounts of BV between all experimental groups. Also, no significant difference was found in the BV, as measured using micro-CT, between the implant groups. Absolute bone ingrowth measurements were highest for collagen-coated implants, but these differences were not significant. CONCLUSION: The obtained data failed to provide a consistent favourable effect on bone formation of the collagen coating over 3 months of implantation. It is concluded that the source of the collagen as well as the limited osseous environment overshadowed a possible effect of the applied implant surface modifications. Similarly, the tested nano-apatite surface coating did not improve peri-implant bone ingrowth into a gap-implant model.

Lateral Bone Augmentation Using a Three-Dimensional-Printed Polymeric Chamber to Compare Biomaterials.

The aim of this study was to test the suitability of calcium phosphate cement mixed with poly(lactic-co-glycolic acid) (CPC-PLGA) microparticles into a ring-shaped polymeric space-maintaining device as bone graft material for lateral bone augmentation. Therefore, the bone chambers were installed on the lateral portion of the anterior region of the mandibular body of mini-pigs. Chambers were filled with either CPC-PLGA or BioOss® particles for comparison and left for 4 and 12 weeks. Histology and histomorphometry were used to obtain temporal insight in material degradation and bone formation. Results indicated that between 4 and 12 weeks of implantation, a significant degradation of the CPC-PLGA (from 75.1% to 23.1%), as well as BioOss material, occurred (from 40.6% to 14.4%). Degradation of both materials was associated with the presence of macrophage-like and osteoclast-like cells. Furthermore, a significant increase in bone formation occurred between 4 and 12 weeks for the CPC-PLGA (from 0.1% to 7.2%), as well as BioOss material (from 8.3% to 23.3%). Statistical analysis showed that bone formation had progressed significantly better using BioOss compared to CPC-PLGA (p < 0.05). In conclusion, this mini-pig study showed that CPC-PLGA does not stimulate lateral bone augmentation using a bone chamber device. Both treatments failed to achieve "clinically" meaningful alveolar ridge augmentation.

Influence of bisphosphonate treatment on bone substitute performance in osteoporotic conditions.

OBJECTIVE: Considering the elevated number of osteoporotic patients in need of bone graft procedures, we here evaluated the effect of alendronate (ALN) treatment on the regeneration of bone defects in osteoporotic rats. Bone formation was histologically and histomorphometrically assessed in rat femoral condyle bone defects filled with bone graft (Bio-Oss®) or left empty. METHODS: Male Wistar rats were induced osteoporotic through orchidectomy (ORX) and SHAM-operated. The animals were divided into three groups: osteoporotic (ORX), osteoporotic treated with ALN (ORX + ALN) and healthy (SHAM). Six weeks after ORX or SHAM surgeries, bone defects were created bilaterally in femoral condyles; one defect was filled with Bio-Oss® and the other one left empty. Bone regeneration within the defects was analyzed by histology and histomorphometry after 4 and 12 weeks. RESULTS: Histological samples showed new bone surrounding Bio-Oss® particles from week 4 onward in all three groups. At week 12, the data further showed that ALN treatment of osteoporotic animals enhanced bone formation to a 10-fold increase compared to non-treated osteoporotic control. Bio-Oss® filling of the defects promoted bone formation at both implantation periods compared to empty controls. CONCLUSION: Our histological and histomorphometric results demonstrate that the enteral administration of alendronate under osteoporotic bone conditions leverages bone defect regeneration to a level comparable to that in healthy bone. Additionally, Bio-Oss® is an effective bone substitute, increasing bone formation, and acting as an osteoconductive scaffold guiding bone growth in both healthy and osteoporotic bone conditions. SIGNIFICANCE: Based on the results of this study, enteral use of ALN mitigates adverse effects of an osteoporotic condition on bone defect regeneration.

Fast Degradable Calcium Phosphate Cement for Maxillofacial Bone Regeneration.

The aim of this preclinical study was to test the applicability of calcium phosphate cement (CPC)-poly(lactic-co-glycolic acid) (PLGA)-carboxymethylcellulose (CMC) as a bone substitute material for guided bone regeneration (GBR) procedures in a clinically relevant mandibular defect model in minipigs. In the study, a predicate device (i.e., BioOss®) was included for comparison. Critical-sized circular mandibular bone defects were created and filled with either CPC-PLGA-CMC without coverage with a GBR membrane or BioOss covered with a GBR membrane and left to heal for 4 and 12 weeks to obtain temporal insight in material degradation and bone formation. Bone formation increased significantly for both CPC-PLGA-CMC and BioOss with increasing implantation time. Further, no significant differences were found for bone formation at either 4 or 12 weeks between CPC-PLGA-CMC and BioOss. Finally, bone substitute material degradation increased significantly for both CPC-PLGA-CMC and BioOss from 4 to 12 weeks of implantation, showing the highest degradation for CPC-PLGA-CMC (∼85%) compared to BioOss (∼12%). In conclusion, this minipig study showed that CPC-PLGA-CMC can be used as a bone-grafting material and stimulates bone regeneration to a comparable extent as with BioOss particles. Importantly, CPC-PLGA-CMC degrades faster compared to BioOss, is easier to apply into a bone defect, and does not need the use of an additional GBR membrane. Consequently, the data support the further investigation of CPC-PLGA-CMC in human clinical trials. Impact statement Guided bone regeneration (GBR) is a frequently used dental surgical technique to regenerate the alveolar ridge to allow stable implant installation. However, stabilization of the GBR membrane and avoidance of bone graft movement remain a challenge. Consequently, there is need for the development of alternative materials to be used in GBR procedures that are easier to apply and induce predictable bone regeneration. In this minipig study, we focused on the applicability of calcium phosphate cement-poly(lactic-co-glycolic acid)-carboxymethylcellulose as an alternative bone substitute material for GBR procedures without the need of an additional GBR membrane.

Comparison of Osteogenic Capacity and Osteoinduction of Adipose Tissue-Derived Cell Populations.

Stromal vascular fraction (SVF) is the primary isolate obtained after enzymatic digestion of adipose tissue that contains various cell types. Its successful application for cell-based construct preparation in an intra-operative setting for clinical bone augmentation and regeneration has been previously reported. However, the performance of SVF-based constructs compared with traditional ex vivo expanded adipose tissue-derived mesenchymal stromal cells (ATMSCs) remains unclear and direct comparative analyses are scarce. Consequently, we here aimed at comparing the in vitro osteogenic differentiation capacity of donor-matched SVF versus ATMSCs as well as their osteoinductive capacity. Human adipose tissue from nine different donors was used to isolate SVF, which was further purified via plastic-adherence to obtain donor-matched ATMSCs. Both cell populations were immunophenotypically characterized for mesenchymal stromal cell, endothelial, and hematopoietic markers after isolation and immunocytochemical staining was used to identify different cell types during prolonged cell culture. Based on normalization using plastic-adherence fraction determination, SVF and ATMSCs were seeded and cultured in osteogenic differentiation medium for 28 days. Further, SVF and ATMSCs were seeded onto devitalized bovine bone granules and subcutaneously implanted into nude mice. After 42 days of implantation, granules were retrieved, histologically processed, and stained with hematoxylin and eosin (HE) to assess ectopic bone formation. The ATMSCs were shown to be a homogenous cell population during cell culture, whereas SVF cultures consisted of multiple cell types. All donor-matched comparisons showed either accelerated or stronger mineralization for SVF cultures in vitro. However, neither SVF nor ATMSCs loaded on devitalized bone granules induced ectopic bone formation on subcutaneous implantation, as opposed to control granules loaded with bone morphogenetic protein-2 (BMP-2), which triggered ectopic bone formation with 100% incidence. Despite the observed lack of osteoinduction, our findings provide important in vitro evidence on the osteogenic superiority of intra-operatively available SVF as compared with donor-matched ATMSCs. Consequently, further studies should focus on optimizing the efficacy of these cell populations for implementation in orthotopic bone fracture or defect treatment.

Cleaved SPP1-rich extracellular vesicles from osteoclasts promote bone regeneration via TGFß1/SMAD3 signaling.

Bone remodeling is a tightly coupled process between bone forming osteoblasts (OBs) and bone resorbing osteoclasts (OCs) to maintain bone architecture and systemic mineral homeostasis throughout life. However, the mechanisms responsible for the coupling between OCs and OBs have not been fully elucidated. Herein, we first validate that secreted extracellular vesicles by osteoclasts (OC-EVs) promote osteogenic differentiation of mesenchymal stem cells (MSCs) and further demonstrate the efficacy of osteoclasts and their secreted EVs in treating tibial bone defects. Furthermore, we show that OC-EVs contain several osteogenesis-promoting proteins as cargo. By employing proteomic and functional analysis, we reveal that mature osteoclasts secrete thrombin cleaved phosphoprotein 1 (SPP1) through extracellular vesicles which triggers MSCs osteogenic differentiation into OBs by activating Transforming Growth Factor ß1 (TGFß1) and Smad family member 3 (SMAD3) signaling. In conclusion, our findings prove an important role of SPP1, present as cargo in OC-derived EVs, in signaling to MSCs and driving their differentiation into OBs. This biological mechanism implies a paradigm shift regarding the role of osteoclasts and their signaling toward the treatment of skeletal disorders which require bone formation.

Repair of complex ovine segmental mandibulectomy utilizing customized tissue engineered bony flaps.

Craniofacial defects require a treatment approach that provides both robust tissues to withstand the forces of mastication and high geometric fidelity that allows restoration of facial architecture. When the surrounding soft tissue is compromised either through lack of quantity (insufficient soft tissue to enclose a graft) or quality (insufficient vascularity or inducible cells), a vascularized construct is needed for reconstruction. Tissue engineering using customized 3D printed bioreactors enables the generation of mechanically robust, vascularized bony tissues of the desired geometry. While this approach has been shown to be effective when utilized for reconstruction of non-load bearing ovine angular defects and partial segmental defects, the two-stage approach to mandibular reconstruction requires testing in a large, load-bearing defect. In this study, 5 sheep underwent bioreactor implantation and the creation of a load-bearing mandibular defect. Two bioreactor geometries were tested: a larger complex bioreactor with a central groove, and a smaller rectangular bioreactor that were filled with a mix of xenograft and autograft (initial bone volume/total volume BV/TV of 31.8 ± 1.6%). At transfer, the tissues generated within large and small bioreactors were composed of a mix of lamellar and woven bone and had BV/TV of 55.3 ± 2.6% and 59.2 ± 6.3%, respectively. After transfer of the large bioreactors to the mandibular defect, the bioreactor tissues continued to remodel, reaching a final BV/TV of 64.5 ± 6.2%. Despite recalcitrant infections, viable osteoblasts were seen within the transferred tissues to the mandibular site at the end of the study, suggesting that a vascularized customized bony flap is a potentially effective reconstructive strategy when combined with an optimal stabilization strategy and local antibiotic delivery prior to development of a deep-seated infection.