An Elastin-derived composite matrix for enhanced vascularized and innervated bone tissue reconstruction: from material development to preclinical evaluation.

Despite progress in bone tissue engineering, reconstruction of large bone defects remains an important clinical challenge. Here, we developed a biomaterial designed to recruit bone cells, endothelial cells, and neuronal fibers within the same matrix, enabling bone tissue regeneration. The bioactive matrix is based on modified elastin-like polypeptides (ELPs) grafted with laminin-derived adhesion peptides IKVAV and YIGSR, and the SNA15 peptide for retention of hydroxyapatite (HA) particles. The composite matrix shows suitable porosity, interconnectivity, biocompatibility for endothelial cells, and the ability to support neurites outgrowth by sensory neurons. Subcutaneous implantation led to the formation of osteoid tissue, characterized by the presence of bone cells, vascular networks, and neuronal structures, while minimizing inflammation. Using a rat femoral condyle defect model, we performed longitudinal micro-CT analysis, which demonstrates a significant increase in the volume of mineralized tissue when using the ELP-based matrix compared to empty defects and a commercially available control (Collapat). Furthermore, visible blood vessel networks and nerve fibers are observed within the lesions after a period of two weeks. By incorporating multiple key components that support cell growth, mineralization, and tissue integration, this ELP-based composite matrix provides a holistic and versatile solution to enhance bone tissue regeneration. This article is protected by copyright. All rights reserved.

Revolutionizing Dental Implant Research: A Systematic Review on Three-Dimensional In Vitro Models.

Dental implants have been clinically used for almost five decades with high success rates. In vitro research models used in implant dentistry are limited to two-dimensional experiments, which are reproducible and well adapted to evaluate a single parameter but do not reproduce the complexity of clinical settings. On the contrary, the in vivo research models using animals offer similar histological and anatomical features to humans, and tissue healing can be close to a clinical situation, but those models are usually accompanied with ethical concerns, and their outcomes could not be extrapolated to humans because of interspecies variabilities. This makes the development of novel in vitro models that recapitulate physiological events occurring during dental implant placement of particular interest for current research in dentistry. Also, such models could be challenged by setting a pathological environment (peri-implantitis) to better understand the disease and eventually serve as a platform to evaluate novel treatment modalities. The aim of this systematic literature review was to cover all the in vitro three-dimensional (3D) complex models available for research in implant dentistry. To accomplish this, a comprehensive search of the literature present on Scopus and PubMed databases was done using specific keywords, as well as inclusion/exclusion criteria. Out of 1334 articles found, we have finally included 27 articles in this review with publication dates between 2001 and 2022. In those articles, the 3D models were designed to study tissue-implant interface behavior in bone or gingival tissue. The articles focused on simulating implant integration, evaluating the effect of different conditions on implant integration, or developing an infection model for the implant integration process. The methods used involved implant material and cells organized in a specific 3D structure. The 3D models developed were able to simulate the process of dental implant osseo- and soft tissue integration and lead to results comparable with conventional in vitro and in vivo models. A relatively limited number of articles were obtained, which indicates that this is an emerging field, highly dependent on progresses made in biotechnologies and tissue engineering, and that further investigation is needed to enhance these 3D in vitro models.

[Uses of 3D printing and Bioprinting for pre-implant bone reconstruction in Oral Surgery]. / Impression 3D et bioimpression pour la régénération osseuse en chirurgie orale.

Pre-implant bone surgery in oral surgery allows to reconstruct maxillary atrophies related to traumatic, infectious or tumoral processes. In this context, the ideal biomaterial remains autogenous bone, but biomaterials (of natural or synthetic origin) allow to limit the morbidity linked to bone harvesting, and to simplify these surgical procedures. In this article, we illustrate how 3D printing technologies can be used as an adjuvant to treat bone defects of complex shape or to create anatomical models used to plan interventions. Finally, some perspectives brought by tissue engineering and bioprinting (creation of complex in vitro models) are presented.

Title: Impression 3D et bioimpression pour la régénération osseuse en chirurgie orale. Abstract: La chirurgie osseuse pré-implantaire en chirurgie orale permet de reconstruire les atrophies des maxillaires en rapport avec des processus traumatiques, infectieux ou tumoraux. Dans ce contexte, le biomatériau idéal reste l'os autogène mais les biomatériaux (d'origine naturelle ou synthétique) permettent de limiter la morbidité liée aux prélèvements osseux et de simplifier ces interventions chirurgicales. Dans cet article, nous illustrons l'apport récent de l'impression 3D dans ce contexte pour traiter des défauts osseux de forme complexe ou pour créer des modèles anatomiques servant à planifier les interventions. Enfin, les perspectives apportées par l'ingénierie tissulaire et la bioimpression (création de modèles in vitro complexes) sont détaillées.

Development of Novel Polysaccharide Membranes for Guided Bone Regeneration: In Vitro and In Vivo Evaluations.

INTRODUCTION: Guided bone regeneration (GBR) procedures require selecting suitable membranes for oral surgery. Pullulan and/or dextran-based polysaccharide materials have shown encouraging results in bone regeneration as bone substitutes but have not been used to produce barrier membranes. The present study aimed to develop and characterize pullulan/dextran-derived membranes for GBR. MATERIALS AND METHODS: Two pullulan/dextran-based membranes, containing or not hydroxyapatite (HA) particles, were developed. In vitro, cytotoxicity evaluation was performed using human bone marrow mesenchymal stem cells (hBMSCs). Biocompatibility was assessed on rats in a subcutaneous model for up to 16 weeks. In vivo, rat femoral defects were created on 36 rats to compare the two pullulan/dextran-based membranes with a commercial collagen membrane (Bio-Gide®). Bone repair was assessed radiologically and histologically. RESULTS: Both polysaccharide membranes demonstrated cytocompatibility and biocompatibility. Micro-computed tomography (micro-CT) analyses at two weeks revealed that the HA-containing membrane promoted a significant increase in bone formation compared to Bio-Gide®. At one month, similar effects were observed among the three membranes in terms of bone regeneration. CONCLUSION: The developed pullulan/dextran-based membranes evidenced biocompatibility without interfering with bone regeneration and maturation. The HA-containing membrane, which facilitated early bone regeneration and offered adequate mechanical support, showed promising potential for GBR procedures.

Association between Dental Scores and Saliva Uremic Toxins.

Dental health is frequently altered in patients with chronic kidney disease. We conducted a prospective study on dental health in CKD patients with a specific interest in the association between dental health issues and the accumulation of uremic toxins in the saliva. A total of 88 patients were included in the study, with chronic kidney disease stage 2 to 5 (without kidney replacement). We analysed the total concentrations of eight uremic toxins (trimethylamine N-oxide -TMAO-, Indoxyl Sulfate, P-cresyl-sulfate, Indole 3-acetic acid, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid -CMPF-, Kynurenine, Hippuric acid and Phenylacetylglutamine) and three precursors of uremic toxins (Tyrosine, Phenylalanine and Tryptophan) in the saliva using LC-MS/MS. We observed, for the first time, the association between various dental scores: DMFT, FST, CPITN, and OHIS, and saliva uremic toxins and precursors: TMAO, indoxyl sulfate, or hippuric acid. Further prospective interventional studies are required to confirm our results.

Oral potentially malignant disorders - An assessment of knowledge and attitude to future education in undergraduate dental students.

INTRODUCTION: The aim of this study was to assess the knowledge and clinical experience of oral potentially malignant disorders (OPMDs) in undergraduate dental students in six European countries (Croatia, France, Italy, Portugal, Spain and United Kingdom) and assess student's attitude and preference to future education on the topic. A secondary aim was to identify gaps in student's knowledge and clinical practice. The study was a part of the Erasmus+ project "Oral Potentially Malignant Disorders: Healthcare Professionals Training" (Grant No: 2020-1-UK01-KA202-078917). MATERIALS AND METHODS: An online questionnaire was distributed to all final-year students in six partner universities. This consisted of four parts assessing: (1) knowledge on OPMDs, (2) clinical experience with this group of patients, (3) self-rated competence in the management of OPMDs and (4) preferences with regard to future education. RESULTS: Two hundred and sixty final-year dental students from six partner universities responded to the questionnaire. Response rates varied from 12% to 92% between partner universities. Significant differences in clinical experience and knowledge were found between students. Students with more clinical exposure to OPMDs rated their knowledge and competence in the management of OPMDs higher than students with less clinical experience. The majority of students were interested in future education on OPMDs, preferably via short educational videos. CONCLUSION: The majority of students have received theoretical knowledge of OPMDs during their undergraduate studies, however, not all had clinical exposure to this group of patients. Students were open to further education on OPMDs. Important deficiencies in knowledge were identified that need to be addressed and it is anticipated that the e-learning platform and e-book that are in development by partner institutions will help to improve overall knowledge of OPMDs.

Malignant Transformation Rate of Oral Submucous Fibrosis: A Systematic Review and Meta-Analysis.

Oral submucous fibrosis (OSF) is a chronic progressive condition affecting the oral cavity, oropharynx and upper third of the oesophagus. It is a potentially malignant disorder. The authors collated and analysed the existing literature to establish the overall malignant transformation rate (MTR). A retrospective analysis of medical and dental scientific literature using online indexed databases was conducted for the period 1956 to 2021. The quality of the enrolled studies was assessed by the Newcastle-Ottawa Scale (NOS). A meta-analysis using a random effects model of a single proportion was performed along with statistical tests for heterogeneity. The overall proportion of malignancy across all studies was 0.06 (95% CI, 0.02-0.10), indicating an overall 6% risk of malignant transformation across all studies and cohorts. Sub-group analyses revealed strong differences in proportion of malignancy according to ethnicity/cohort; Chinese = 0.02 (95% CI 0.01-0.02), Taiwanese = 0.06 (95% CI, 0.03-0.10), Indian = 0.08 (95% CI, 0.03-0.14) and Pakistani = 0.27 (95% CI 0.25-0.29). Overall, the MTR was 6%; however, wide heterogeneity of the included studies was noted. Geographic variations in MTR were noted but were not statistically significant. Further studies are required to analyse the difference between cohort groups.

Accuracy of commercial 3D printers for the fabrication of surgical guides in dental implantology.

OBJECTIVES: To evaluate the accuracy of two different surgical guides (small extent = single implant and large extent = full arch) fabricated by five additive manufacturing technologies (SLA=Stereolithography, DLP= Digital Light Processing, FDM=Fused Deposition Modeling, SLS=Selective Laser Sintering, Inkjet). METHODS: Overall, 72 guides (6 per type) were obtained with the different machines (SLA=Form2; DLP=Rapid Shape D40 and Cara Print 4.0; FDM=Raise 3D Pro2; SLS=Prodways P1000; Polyjet®=Stratasys J750). The guides were surface-scanned with an optical dental scanner, and the resulting files were compared with the initial design files using a surface matching software. Root Mean Square (RMS) and standard deviation were calculated, representing respectively trueness and precision. Kruskall-Wallis non-parametric test was used to compare trueness and precision between small-extent and large-extent guides and 3D printer by pairs. The threshold for significance was α=0.05, except for the comparison of printers by pairs where a Bonferroni-corrected level of 0.0033 was used. RESULTS: Significant differences were observed for trueness and precision between small-extent and large-extent guides, regardless the printer except for DLP (trueness and precision) and SLS (precision). SLA, DLP and Polyjet® technologies showed similar results in terms of trueness and precision for both small-extend and large-extend guides (P>0.05). CONCLUSIONS: The size affected the accuracy of CAD-CAM surgical guides. The different additive manufacturing technologies had a limited impact on the accuracy. CLINICAL SIGNIFICANCE: This study is of clinical interest as it shows that the 3D printing technology (SLA/DLP) has a limited impact on 3D printed surgical guides accuracy. However, the size of the guide can have a significant impact, as small-extent guides were more accurate than large-extent guides.

Applications of Human Amniotic Membrane for Tissue Engineering.

An important component of tissue engineering (TE) is the supporting matrix upon which cells and tissues grow, also known as the scaffold. Scaffolds must easily integrate with host tissue and provide an excellent environment for cell growth and differentiation. Human amniotic membrane (hAM) is considered as a surgical waste without ethical issue, so it is a highly abundant, cost-effective, and readily available biomaterial. It has biocompatibility, low immunogenicity, adequate mechanical properties (permeability, stability, elasticity, flexibility, resorbability), and good cell adhesion. It exerts anti-inflammatory, antifibrotic, and antimutagenic properties and pain-relieving effects. It is also a source of growth factors, cytokines, and hAM cells with stem cell properties. This important source for scaffolding material has been widely studied and used in various areas of tissue repair: corneal repair, chronic wound treatment, genital reconstruction, tendon repair, microvascular reconstruction, nerve repair, and intraoral reconstruction. Depending on the targeted application, hAM has been used as a simple scaffold or seeded with various types of cells that are able to grow and differentiate. Thus, this natural biomaterial offers a wide range of applications in TE applications. Here, we review hAM properties as a biocompatible and degradable scaffold. Its use strategies (i.e., alone or combined with cells, cell seeding) and its degradation rate are also presented.

Comparison of amniotic membrane versus the induced membrane for bone regeneration in long bone segmental defects using calcium phosphate cement loaded with BMP-2.

Thanks to its biological properties, the human amniotic membrane (HAM) combined with a bone substitute could be a single-step surgical alternative to the two-step Masquelet induced membrane (IM) technique for regeneration of critical bone defects. However, no study has directly compared these two membranes. We first designed a 3D-printed scaffold using calcium phosphate cement (CPC). We assessed its suitability in vitro to support human bone marrow mesenchymal stromal cells (hBMSCs) attachment and osteodifferentiation. We then performed a rat femoral critical size defect to compare the two-step IM technique with a single-step approach using the HAM. Five conditions were compared. Group 1 was left empty. Group 2 received the CPC scaffold loaded with rh-BMP2 (CPC/BMP2). Group 3 and 4 received the CPC/BMP2 scaffold covered with lyophilized or decellularized/lyophilized HAM. Group 5 underwent a two- step induced membrane procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by, after 4 weeks, its replacement with the CPC/BMP2 scaffold wrapped in the IM. Micro-CT and histomorphometric analysis were performed after six weeks. Results showed that the CPC scaffold supported the proliferation and osteodifferentiation of hBMSCs in vitro. In vivo, the CPC/BMP2 scaffold very efficiently induced bone formation and led to satisfactory healing of the femoral defect, in a single-step, without autograft or the need for any membrane covering. In this study, there was no difference between the two-step induced membrane procedure and a single step approach. However, the results indicated that none of the tested membranes further enhanced bone healing compared to the CPC/BMP2 group.

Bone regeneration in both small and large preclinical bone defect models using an injectable polymer-based substitute containing hydroxyapatite and reconstituted with saline or autologous blood.

Microbeads consisting of pullulan and dextran supplemented with hydroxyapatite have recently been developed for bone tissue engineering applications. Here, we evaluate the bone formation in two different preclinical models after injection of microbeads reconstituted with either saline buffer or autologous blood. Addition of saline solution or autologous blood to dried microbeads packaged into syringes allowed an easy injection. In the first rat bone defect model performed in the femoral condyle, microcomputed tomography performed after 30 and 60 days revealed an important mineralization process occurring around and within the core of the microbeads in both conditions. Bone volume/total volume measurements revealed no significant differences between the saline solution and the autologous blood groups. Histologically, osteoid tissue was evidenced around and in contact of the microbeads in both conditions. Using the sinus lift model performed in sheep, cone beam computed tomography revealed an important mineralization inside the sinus cavity for both groups after 3 months of implantation. Representative Masson trichrome staining images showed that bone formation occurs at the periphery and inside the microbeads in both conditions. Quantitative evaluation of the new bone formation displayed no significant differences between groups. In conclusion, reconstitution of microbeads with autologous blood did not enhance the regenerative capacity of these microbeads compared to the saline buffer group. This study is of particular interest for clinical applications in oral and maxillofacial surgery.

Development and characterization of a PLGA-HA composite material to fabricate 3D-printed scaffolds for bone tissue engineering.

Additive manufacturing is a rising field in bone tissue engineering. Additive fabrication offers reproducibility, high precision and rapid manufacture of custom patient-specific scaffolds. The development of appropriate composite materials for biomedical applications is critical to reach clinical application of these novel biomaterials. In this work, medical grade poly(lactic-co-glycolic) acid (PLGA) was mixed with hydroxyapatite nanoparticles (nHA) to fabricate 3D porous scaffolds by Fused Deposition Modeling. We have first confirmed that the composite material could be printed in a reproductive manner. Physical characterization demonstrated a low degradation of the material during manufacturing steps and an expected loading and homogeneous distribution of nHA. In vitro biodegradation of the scaffolds showed modifications of morphological and physicochemical properties over time. The composite scaffolds were biocompatible and high cell viability was observed in vitro, as well as a maintain of cell proliferation. As expected, the addition of nHA displayed a positive impact on osteodifferentiation in vitro. Furthermore, a limited inflammatory reaction was observed after subcutaneous implantation of the materials in the rat. Overall, this study suggests that this composite material is suitable for bone tissue engineering applications.

Chorion and amnion/chorion membranes in oral and periodontal surgery: A systematic review.

The aim of this study was to perform a systematic review on the clinical applications where chorion membrane (CM) and amnion/chorion membrane (ACM) were used for oral tissue regeneration procedures. Selection of articles was carried out by two evaluators in Pubmed and Scopus databases, and Outcomes (PICO) method was used to select the relevant articles. Clinical studies reporting the use of CM or ACM for oral soft and hard tissue regeneration were included. The research involved 21 studies conducted on 375 human patients. Seven clinical applications of CM and ACM in oral and periodontal surgery were identified: gingival recession treatment, intrabony and furcation defect treatment, alveolar ridge preservation, keratinized gum width augmentation around dental implants, maxillary sinus membrane repair, and large bone defect reconstruction. CM and ACM were compared to negative controls (conventional surgeries without membrane) or to the following materials: collagen membranes, dense polytetrafluoroethylene membranes, platelet-rich fibrin membranes, amnion membranes, and to a bone substitute. Several studies support the use of CM and ACM as an efficient alternative to current techniques for periodontal and oral soft tissue regeneration procedures. However, further studies are necessary to increase the level of evidence and especially to demonstrate their role for bone regeneration.

Correction to: 3D printed bone models in oral and craniomaxillofacial surgery: a systematic review.

An amendment to this paper has been published and can be accessed via the original article.

A Novel Xenograft Bone Substitute Supports Stable Bone Formation in Circumferential Defects Around Dental Implants in Minipigs.

PURPOSE: The aim of this study was to evaluate and compare bone growth and implant integration in circumferential defects with two commercially available bone substitutes (demineralized bovine bone mineral [DBBM]). MATERIALS AND METHODS: Circumferential defects were created in the mandibles of minipigs (n = 10), and Bone Level Tapered implants (Straumann Roxolid with SLActive surface) were placed. The defects (4-mm-deep circumferential defect, 2 mm around each implant) were augmented with either sintered bovine bone mineral (test, cerabone) or natural bovine bone mineral (control, Bio-Oss). Bone formation and tissue composition in augmented sites were histomorphometrically assessed after 8 and 12 weeks of healing time (n = 5 each), respectively, in terms of the percentage of area of newly formed bone to total area, bone-to-implant contact (BIC), and crestal bone height relative to the implant shoulder (first bone-to-implant contact [fBIC]). RESULTS: Bone formation in all defect sites was adequate and equivalent for both groups at individual healing time points. The amount of residual graft material was comparable in both groups after 8 and 12 weeks, with no significant resorption in either group. The mean newly formed bone area in the test group amounted to 46.7% ± 5.1% and 48.7% ± 4.0% after 8 and 12 weeks vs 47.0% ± 4.8% and 47.8% ± 7.3% in the control group, respectively. BIC and fBIC as individually assessed for the lingual and buccal aspects were comparable at both healing time points without any statistically significant differences between the groups. A slightly greater variability of fBIC was observed within the test group. CONCLUSION: The results of this study indicate that test and control materials both represent viable bovine bone graft material that equivalently support the formation of new and stable bone volume specifically when used for simultaneous augmentation around implants.

3D printed bone models in oral and cranio-maxillofacial surgery: a systematic review.

AIM: This systematic review aimed to evaluate the use of three-dimensional (3D) printed bone models for training, simulating and/or planning interventions in oral and cranio-maxillofacial surgery. MATERIALS AND METHODS: A systematic search was conducted using PubMed® and SCOPUS® databases, up to March 10, 2019, by following the Preferred Reporting Items for Systematic reviews and Meta-Analysis (PRISMA) protocol. Study selection, quality assessment (modified Critical Appraisal Skills Program tool) and data extraction were performed by two independent reviewers. All original full papers written in English/French/Italian and dealing with the fabrication of 3D printed models of head bone structures, designed from 3D radiological data were included. Multiple parameters and data were investigated, such as author's purpose, data acquisition systems, printing technologies and materials, accuracy, haptic feedback, variations in treatment time, differences in clinical outcomes, costs, production time and cost-effectiveness. RESULTS: Among the 1157 retrieved abstracts, only 69 met the inclusion criteria. 3D printed bone models were mainly used as training or simulation models for tumor removal, or bone reconstruction. Material jetting printers showed best performance but the highest cost. Stereolithographic, laser sintering and binder jetting printers allowed to create accurate models with adequate haptic feedback. The cheap fused deposition modeling printers exhibited satisfactory results for creating training models. CONCLUSION: Patient-specific 3D printed models are known to be useful surgical and educational tools. Faced with the large diversity of software, printing technologies and materials, the clinical team should invest in a 3D printer specifically adapted to the final application.

Alendronate or Zoledronic acid do not impair wound healing after tooth extraction in postmenopausal women with osteoporosis.

BACKGROUND: Bisphosphonates (BPs) are widely used for the prevention or treatment of osteoporosis. One of the most serious complications associated with BPs is medication-related osteonecrosis of the jaw (MRONJ) but its incidence in patients with osteoporosis is very low ranging from 0.001-0.15%. A major predisposing factor for MRONJ is tooth extraction (TE). Controversies persist about the influence of current BP therapy regarding socket healing after TE. The aims of this study were to investigate prospectively, (i) alveolar bone healing, i.e., filling of the bony socket by new bone and (ii) mucosal healing, i.e., closure of the overlying mucosa, after TE in women receiving current BP therapy for the prevention or the treatment of postmenopausal osteoporosis. METHODS: Women with osteoporosis under current treatment with BPs (BP+ group) or other anti-osteoporotic medications (BP- group) undergoing single TE were included in this study. No antibiotic prophylaxis was prescribed solely for the BP therapy, but antibiotic treatment may have been required for local infectious conditions. Chlorohexidine mouthwashes were systematically prescribed in all study patients for one week after TE. New bone height (NBH) and rate of socket filling (RSF) were recorded using intraoral standardized radiographs one month and 3 months after TE (T30 and T90 respectively). The closure of the overlying mucosa was assessed by measuring the wound extent with an electronic caliper at 1 week and at 1 month after TE (T7 and T30 respectively). RESULTS: At T30, NBH was not statistically different between the BP+ and BP- groups (p = .76). At T90, more than a two-fold in NBH increase was recorded for both groups with no statistically significant difference between them (p = .76). At T30 and T90, RSF was similar in both groups (p = .58 and p = .32 respectively). More than a two-fold RSF increase was founded between T30 and T90 in both groups. No demographic or BPs-related factors were correlated with the RSF at T90. At T7, the mucosa wound extent was reduced by more than two-fold with no statistically significant difference between both groups (p = .80). At this time, mucosa healing was achieved in 11.9% of the BP+ group and 10% of the BP- group (p = .99). At T30, mucosal healing was achieved in all patients but two, and at T90 it was achieved in all patients. CONCLUSION: This study provides new insights into bone and mucosal healing in patients with osteoporosis taking BPs after TE. In this population, TE can be managed successfully with an appropriate surgical protocol and without discontinuation of BP treatment.

Assessment of fresh and preserved amniotic membrane for guided bone regeneration in mice.

Thanks to its biological properties, the human amniotic membrane (HAM) can be used as a barrier membrane for guided bone regeneration (GBR). However, no study has assessed the influence of the preservation method of HAM for this application. This study aimed to establish the most suitable preservation method of HAM for GBR. Fresh (F), cryopreserved (C) lyophilized (L), and decellularized and lyophilized (DL) HAM were compared. The impact of preservation methods on collagen and glycosaminoglycans (GAG) content was evaluated using Masson's trichrome and alcian blue staining. Their suture retention strengths were assessed. In vitro, the osteogenic potential of human bone marrow mesenchymal stromal cells (hBMSCs) cultured on the four HAMs was evaluated using alkaline phosphatase staining and alizarin red quantification assay. In vivo, the effectiveness of fresh and preserved HAMs for GBR was assessed in a mice diaphyseal bone defect after 1 week or 1 month healing. Micro-CT and histomorphometric analysis were performed. The major structural components of HAM (collagen and GAG) were preserved whatever the preservation method used. The tearing strength of DL-HAM was significantly higher. In vitro, hBMSCs seeded on DL-HAM displayed a stronger ALP staining, and alizarin red staining quantification was significantly higher at Day 14. In vivo, L-HAM and DL-HAM significantly enhanced early bone regeneration. One month after the surgery, only DL-HAM slightly promoted bone regeneration. Several preserving methods of HAM have been studied for bone regeneration. Here, we have demonstrated that DL-HAM achieved the most promising results for GBR.

Comparison of the impact of preservation methods on amniotic membrane properties for tissue engineering applications.

Human amniotic membrane (hAM) is considered as an attractive biological scaffold for tissue engineering. For this application, hAM has been mainly processed using cryopreservation, lyophilization and/or decellularization. However, no study has formally compared the influence of these treatments on hAM properties. The aim of this study was to develop a new decellularization-preservation process of hAM, and to compare it with other conventional treatments (fresh, cryopreserved and lyophilized). The hAM was decellularized (D-hAM) using an enzymatic method followed by a detergent decellularization method, and was then lyophilized and gamma-sterilized. Decellularization was assessed using DNA staining and quantification. D-hAM was compared to fresh (F-hAM), cryopreserved (C-hAM) and lyophilized/gamma-sterilized (L-hAM) hAM. Their cytotoxicity on human bone marrow mesenchymal stem cells (hBMSCs) and their biocompatibility in a rat subcutaneous model were also evaluated. The protocol was effective as judged by the absence of nuclei staining and the residual DNA lower than 50 ng/mg. Histological staining showed a disruption of the D-hAM architecture, and its thickness was 84% lower than fresh hAM (p < 0.001). Despite this, the labeling of type IV and type V collagen, elastin and laminin were preserved on D-hAM. Maximal force before rupture of D-hAM was 92% higher than C-hAM and L-hAM (p < 0.01), and D-hAM was 37% more stretchable than F-hAM (p < 0.05). None of the four hAM were cytotoxic, and D-hAM was the most suitable scaffold for hBMSCs proliferation. Finally, D-hAM was well integrated in vivo. In conclusion, this new hAM decellularization process appears promising for tissue engineering applications.

Layer-by-layer bioassembly of poly(lactic) acid membranes loaded with coculture of HBMSCs and EPCs improves vascularization in vivo.

Layer-by-layer (LBL) BioAssembly method was developed to enhance the control of cell distribution within 3D scaffolds for tissue engineering applications. The objective of this study was to evaluate in vivo the development of blood vessels within LBL bioassembled membranes seeded with human primary cells, and to compare it to cellularized massive scaffolds. Poly(lactic) acid (PLA) membranes fabricated by fused deposition modeling were seeded with monocultures of human bone marrow stromal cells or with cocultures of these cells and endothelial progenitor cells. Then, four cellularized membranes were assembled in LBL constructs. Early osteoblastic and endothelial cell differentiation markers, alkaline phosphatase, and von Willebrand's factor, were expressed in all layers of assemblies in homogenous manner. The same kind of LBL assemblies as well as cellularized massive scaffolds was implanted subcutaneously in mice. Human cells were observed in all scaffolds seeded with cells, but not in the inner parts of massive scaffolds. There were significantly more blood vessels observed in LBL bioassemblies seeded with cocultures compared to all other samples. LBL bioassembly of PLA membranes seeded with a coculture of human cells is an efficient method to obtain homogenous cell distribution and blood vessel formation within the entire volume of a 3D composite scaffold.