In vitro osteodifferentiation of intact human amniotic membrane is not beneficial in the context of bone repair.

The human amniotic membrane (hAM) is an attractive biomaterial for regenerative medicine, as it contains amniotic mesenchymal stromal cells (hAMSC), epithelial cells (hAEC) and growth factors. We examined the potential use of hAM in orthopaedic and maxillofacial bone surgery, integrating the requirements of current regulations regarding advanced therapy medicinal products (ATMP) in the European Union. Previous studies have described the potential osteodifferentiation of intact hAM during whole-tissue culture in osteogenic conditions. The present study aims to determine whether in vitro osteodifferentiation of hAM is needed in the context bone repair, and the influence of this process on tissue structure, cell phenotype and cell function. Different conditions (fresh or cultured hAM; intact or hAM-derived cells) were tested. Phenotypic and functional analyses were performed with standard approaches (cell culture and staining, histological and immunolabelling) as well as original approaches (tissue staining, energy dispersive X-ray and X-ray diffraction). In our study, non-osteodifferentiated hAM (i.e., fresh or native hAM) exhibited innate pre-osteoblastic potential. Osteodifferentiation of fresh hAM induced a change in tissue structure, cell phenotype and function. Therefore, we hypothesize that pre-osteodifferentiation may not be necessary, especially if it induces unwanted changes. To our surprise, in these osteogenic conditions, hAEC had a mesenchymal phenotype with osteocyte function, and even native synthesis of hydroxyapatite, focusing osteogenic potential mainly in this epithelial layer. In conclusion, in vitro osteodifferentiation by tissue culture does not appear to be necessary for hAM to be used as an innovative ATMP for bone repair.

Human amniotic membrane for guided bone regeneration of calvarial defects in mice.

Due to its biological properties, human amniotic membrane (hAM) is widely studied in the field of tissue engineering and regenerative medicine. hAM is already very attractive for wound healing and it may be helpful as a support for bone regeneration. However, few studies assessed its potential for guided bone regeneration (GBR). The purpose of the present study was to assess the potential of the hAM as a membrane for GBR. In vitro, cell viability in fresh and cryopreserved hAM was assessed. In vivo, we evaluated the impact of fresh versus cryopreserved hAM, using both the epithelial or the mesenchymal layer facing the defect, on bone regeneration in a critical calvarial bone defect in mice. Then, the efficacy of cryopreserved hAM associated with a bone substitute was compared to a collagen membrane currently used for GBR. In vitro, no statistical difference was observed between the conditions concerning cell viability. Without graft material, cryopreserved hAM induced more bone formation when the mesenchymal layer covered the defect compared to the defect left empty. When associated with a bone substitute, such improved bone repair was not observed. These preliminary results suggest that cryopreserved hAM has a limited potential for GBR.

Fresh and in vitro osteodifferentiated human amniotic membrane, alone or associated with an additional scaffold, does not induce ectopic bone formation in Balb/c mice.

The human amniotic membrane (hAM) has been successfully used as a natural carrier containing amniotic mesenchymal stromal cells, epithelial cells and growth factors. It has a little or no immunogenicity, and possesses useful anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. It has been used for many years in several indications for soft tissue repair. We previously reported that hAM represents a natural and preformed sheet containing highly potent stem cells, and could thus be used for bone repair. Indeed, native hAM possesses pre-osteoblastic potential that can easily be stimulated, even as far as mineralization, by means of in vitro osteogenic culture. However, cell culture induces damage to the tissue, as well as to cell phenotype and function. The aim of this study was to evaluate new bone formation by fresh and in vitro osteodifferentiated hAM, alone or associated with an additional scaffold presenting osteoinductive properties. Moreover, we also aimed to determine the effect of in vitro hAM pre-osteodifferentiation on its in vivo biocompatibility/tissue degradation. Results showed that neither fresh nor osteodifferentiated hAM induced ectopic bone formation, whether or not it was associated with the osteoinductive scaffold. Secondly, fresh and osteodifferentiated hAM presented similar in vivo tissue degradation, suggesting that in vitro hAM pre-osteodifferentiation did not influence its in vivo biocompatibility.

Storage and qualification of viable intact human amniotic graft and technology transfer to a tissue bank.

Human amniotic membrane (hAM) is known to have good potential to help the regeneration of tissue. It has been used for over 100 years in many medical disciplines because of its properties, namely a scaffold containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. In order to use this "boosted membrane" as an advanced therapeutic medicinal product for bone repair, we aimed to observe the influence of tissue culture and/or cryopreservation on cell viability and tissue structure, and secondly, to adapt to a tissue bank, identify easy processes to store hAM containing viable cells and to verify the quality of the graft before its release for use. To this end, we tested different published culture or cryopreservation storage conditions and cell viability assays. Tissue structure was evaluated by Giemsa staining and was compared to histological analysis. Preliminary results show no dramatic decrease in cell viability in cultured hAM as compared to cryopreserved hAM, but tissue structure alterations were observed with both storage conditions. Histological and immunohistochemical data highlight that tissue damage was associated with significantly modified protein expression, which could lead to a possible loss of differentiation potential. Finally, we report that trypan blue and Giemsa staining could constitute controls that are "materially and easily transferable" to a tissue bank.

Expert Revision of Key Elements for Clinical-Grade Production and Qualification of Perinatal Derivatives.

Perinatal derivatives have been proposed as adjunct therapeutic strategies or innovative treatments. Undoubtedly, perinatal derivatives can offer the opportunity and source material to isolate multipotent stem cells, but both maternal- and fetal-derived tissues can be processed and transformed into engineered tissues or advanced biomedical devices, whose potential remains to be fully elucidated. Promising preclinical and clinical results collected so far clearly foresee an escalation of such novel treatments. Market forecasts predict exponential growth in such advanced medicinal products during the next decade, with a pragmatic innovation for medicine into a more advanced biomedical version, enlarging the portfolio for treating a wide range of congenital and acute conditions. However, all these promising and fascinating therapeutic possibilities cannot gain a solid and recognized role in established medical practice without rigid and harmonized manufacturing strategies. The implementation of strategies according to guidelines and directives compiled by Regulatory Agencies, in conformity to (European) Pharmacopoeia and for Good Manufacturing Practice -conforming production of such products, represent critical steps required to translate perinatal technologies into effective therapeutic approaches. During the past 5 years, a panel of European experts and developers, gathered under the umbrella of the COST Sprint Action, supported by the European Cooperation in Science and Technology action, had the opportunity to revise and summarize experience and recommendations for a fruitful and proficient generation of perinatal biomedical products. In order to facilitate the creation and potential commercialization of perinatal bioengineered and advanced pharmaceutical products and technologies, such a collection of data and recommendations is described and discussed here.

Questions about Using the Induced Membrane Technique to Manage Cases of Congenital Tibial Pseudarthrosis.

The induced membrane technique is an innovative approach for repairing critical bone defects and has been applied recently in patients with congenital pseudarthrosis of the tibia (CPT). CPT is frequently associated with neurofibromatosis type 1 (NF1). Here, we briefly describe the clinical results of the induced membrane technique in NF1-deficient patients with CPT and in an animal model of CPT. Furthermore, we discuss the hypotheses used to explain inconsistent outcomes for the induced membrane technique in CPT-especially when associated with NF1.

Mapping of the Human Amniotic Membrane: In Situ Detection of Microvesicles Secreted by Amniotic Epithelial Cells.

The potential clinical applications of human amniotic membrane (hAM) and human amniotic epithelial cells (hAECs) in the field of regenerative medicine have been known in literature since long. However, it has yet to be elucidated whether hAM contains different anatomical regions with different plasticity and differentiation potential. Recently, for the first time, we highlighted many differences in terms of morphology, marker expression, and differentiation capabilities among four distinct anatomical regions of hAM, demonstrating peculiar functional features in hAEC populations. The aim of this study was to investigate in situ the ultrastructure of the four different regions of hAM by means of transmission electron microscopy (TEM) to deeply understand their peculiar characteristics and to investigate the presence and localization of secretory products because to our knowledge, there are no similar studies in the literature. The results of this study confirm our previous observations of hAM heterogeneity and highlight for the first time that hAM can produce extracellular vesicles (EVs) in a heterogeneous manner. These findings should be considered to increase efficiency of hAM applications within a therapeutic context.

Expert Consideration on Regulatory Aspects for Perinatal Derivatives in Clinical Settings.

Perinatal derivatives (PnD) are drawing growing interest among the scientific community as an unrestricted source of multipotent stem cells, secretome, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options, but they require the development of regenerative approaches. With this development, the question of regulation of donation, processing, and distribution has therefore become more important. Within the European Cooperation in Science and Technology (COST) community, we compiled a group of international experts on PnD technologies, who revised and compared existing EU national regulations. Notably, despite clear European directives, each EU Country has developed their own implementation and standard levels for cell- and tissue-based therapies. To enable extended applications of PnD treatments within the EU community and worldwide, harmonization is highly recommended. This paper aims to provide an overview of the various options available to introduce PnD into clinical practice. For this purpose, the different aspects resulting from (1) the type of PnD, (2) the amount of available data, (3) the degree of manipulation, and (4) the intended application and the process toward a possible commercialization will be presented. In the future, it will be important to find a balance between regulatory requirements and the best medical quality of the PnD product.

Questions about Residual Cell Viability in Cryopreserved Human Amniotic Membrane and Its Impact on Clinical Applications.

We questioned the relevance of evaluating residual cell viability in human amniotic membrane (hAM) after its cryopreservation since cell survival is controversial and its ability to act as a matrix (including the presence of growth factors and cytokines) appears to be most important for tissue regeneration purposes. We also discussed the usefulness of osteodifferentiating amniotic cells in whole hAM for bone repair applications. We have evidence that determining residual cell viability after cryopreservation and hAM osteodifferentiation is not justified.

New Devitalized Freeze-Dried Human Umbilical Cord Amniotic Membrane as an Innovative Treatment of Ocular Surface Defects: Preclinical Results.

A preclinical study was performed to investigate the efficacy and safety of a new viral inactivated, devitalized, freeze-dried and gamma-sterilized human umbilical cord amniotic membrane (lhUC-AM) for the treatment of deep scleral and corneal defects with or without perforation. Firstly, lhUC-AM was investigated on experimental deep sclerectomy in rabbit eyes (n = 12) and compared to autograft (n = 4) on cross section histology. Secondly, lhUC-AM was studied on a selected series of uncontrolled cases of corneal defects (n = 18) with or without perforation, in dogs and cats. lhUC-AM tolerance, reconstruction of the deep corneal lesion and recovery of the structural aspect of the tissue were followed post-surgery. In experimental deep sclerectomy, histology showed that the lhUC-AM was well tolerated and degraded completely in 45 days while allowing an overall quality and kinetic of scleral regeneration, similar to autograft. In the clinical situations, lhUC-AM was well tolerated, with ocular inflammatory signs quickly decreasing after surgery. Mean follow-up was 16.40 ± 11.43 months. In 15 out of 18 cases, lhUC-AM allowed ocular surface wound healing. The ocular surface was fully reconstructed three months after surgery. This study suggests a good safety and efficacy profile of lhUC-AM in the treatment of deep corneal or scleral defect in animals. This new tissue should now facilitate the treatment of severe ocular surface diseases in humans.

Development of a biomimetic bioreactor for regenerative endodontics research.

In the context of regenerative endodontics research with the development of biomaterials, this work aimed to develop and test a prototype biomimetic bioreactor of a human tooth. The bioreactor was designed to reproduce a shaped dental canal connected with a cavity reproducing the periapical region and irrigated through two fluidic channels intended to reproduce the apical residual vascular supply. A test biomaterial composed of polylactic acid/polycaprolactone-tannic acid (PLA/PCL-TA) was produced by electrospinning/electrospraying and calibrated to be inserted in a dental canal. This biomaterial was first used to evaluate its imbibition capacity and the oximetry inside the bioreactor. Then, Dental Pulp Stem Cells (DPSCs) were cultured on PLA/PCL-TA cones for 1-3 weeks in the bioreactor; afterward cell adhesion, proliferation, and migration were histologically assessed. Complete imbibition biomaterial was obtained in 10 min and oximetry was stable over time. In the bioreactor, DPSCs were able to adhere, proliferate and migrate onto the surface and inside the biomaterial. In conclusion, this bioreactor was used successfully to test a biomaterial intended to support pulp regeneration and constitutes a new in vitro experimental model closer to clinical reality.

Antibacterial and Immunomodulatory Properties of Acellular Wharton's Jelly Matrix.

Of all biologic matrices, decellularized tissues have emerged as a promising tool in the field of regenerative medicine. Few empirical clinical studies have shown that Wharton's jelly (WJ) of the human umbilical cord promotes wound closure and reduces wound-related infections. In this scope, we herein investigated whether decellularized (DC)-WJ could be used as an engineered biomaterial. In comparison with devitalized (DV)-WJ, our results showed an inherent effect of DC-WJ on Gram positive (S. aureus and S. epidermidis) and Gram negative (E. coli and P. aeruginosa) growth and adhesion. Although DC-WJ activated the neutrophils and monocytes in a comparable magnitude to DV-WJ, macrophages modulated their phenotypes and polarization states from the resting M0 phenotype to the hybrid M1/M2 phenotype in the presence of DC-WJ. M1 phenotype was predominant in the presence of DV-WJ. Finally, the subcutaneous implantation of DC-WJ showed total resorption after three weeks of implantation without any sign of foreign body reaction. These significant data shed light on the potential regenerative application of DC-WJ in providing a suitable biomaterial for tissue regenerative medicine and an ideal strategy to prevent wound-associated infections.

Human amniotic membrane application in oral surgery-An ex vivo pilot study.

Objectives: The purpose of this pilot porcine study was to explore and illustrate the surgical application of human amniotic membrane (hAM) in an ex vivo model of medication-related osteonecrosis of the jaw (MRONJ). Material and methods: Five oral and maxillofacial surgeons participated to this study. MRONJ was simulated on porcine mandible specimens. hAM was applied using four different techniques: implantation with complete coverage, implantation with partial coverage, apposition and covering graft material. At the same time, the surgeons evaluated how well the hAM handled and its physical properties during the surgery. Results: Surgeons found that hAM had suitable mechanical properties, as it was easy to detach from the support, handle, bind to the defect and bury. hAM was also found to be strong and stable. The "implantation with complete coverage" and "implantation with partial coverage" techniques were the preferred choices for the MRONJ indication. Conclusion: This study shows that hAM is a graft material with suitable properties for oral surgery. It is preferable to use it buried under the gingiva with sutures above it, which increases its stability. This technical note aims to educate surgeons and provide them with details about the handling of hAM in oral surgery. Clinical relevance: Two surgical techniques for hAM application in MRONJ were identified and illustrated. hAM handling and physical properties during surgery were reported.

Perinatal derivatives: How to best characterize their multimodal functions in vitro. Part C: Inflammation, angiogenesis, and wound healing.

Perinatal derivatives (PnD) are birth-associated tissues, such as placenta, umbilical cord, amniotic and chorionic membrane, and thereof-derived cells as well as secretomes. PnD play an increasing therapeutic role with beneficial effects on the treatment of various diseases. The aim of this review is to elucidate the modes of action of non-hematopoietic PnD on inflammation, angiogenesis and wound healing. We describe the source and type of PnD with a special focus on their effects on inflammation and immune response, on vascular function as well as on cutaneous and oral wound healing, which is a complex process that comprises hemostasis, inflammation, proliferation (including epithelialization, angiogenesis), and remodeling. We further evaluate the different in vitro assays currently used for assessing selected functional and therapeutic PnD properties. This review is a joint effort from the COST SPRINT Action (CA17116) with the intention to promote PnD into the clinics. It is part of a quadrinomial series on functional assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer activities, anti-inflammation, wound healing, angiogenesis, and regeneration.

Methods and criteria for validating the multimodal functions of perinatal derivatives when used in oncological and antimicrobial applications.

Perinatal derivatives or PnDs refer to tissues, cells and secretomes from perinatal, or birth-associated tissues. In the past 2 decades PnDs have been highly investigated for their multimodal mechanisms of action that have been exploited in various disease settings, including in different cancers and infections. Indeed, there is growing evidence that PnDs possess anticancer and antimicrobial activities, but an urgent issue that needs to be addressed is the reproducible evaluation of efficacy, both in vitro and in vivo. Herein we present the most commonly used functional assays for the assessment of antitumor and antimicrobial properties of PnDs, and we discuss their advantages and disadvantages in assessing the functionality. This review is part of a quadrinomial series on functional assays for the validation of PnDs spanning biological functions such as immunomodulation, anticancer and antimicrobial, wound healing, and regeneration.

Perinatal derivatives application: Identifying possibilities for clinical use.

Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.

General consensus on multimodal functions and validation analysis of perinatal derivatives for regenerative medicine applications.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration.

Reporting Criteria for Clinical Trials on Medication-Related Osteonecrosis of the Jaw (MRONJ): A Review and Recommendations.

Medication-related osteonecrosis of the jaw (MRONJ) is a complication caused by anti-resorptive agents and anti-angiogenesis drugs. Since we wanted to write a protocol for a randomized clinical trial (RCT), we reviewed the literature for the essential information needed to estimate the size of the active patient population and measure the effects of therapeutics. At the same time, we designed a questionnaire intended for clinicians to collect detailed information about their practices. Twelve essential criteria and seven additional items were identified and compiled from 43 selected articles. Some of these criteria were incorporated in the questionnaire coupled with data on clinical practices. Our review found extensive missing data and a lack of consensus. For example, the success rate often combined MRONJ stages, diseases, and drug treatments. The occurrence date and evaluation methods were not harmonized or quantitative enough. The primary and secondary endpoints, failure definition, and date coupled to bone measurements were not well established. This information is critical for writing a RCT protocol. With this review article, we aim to encourage authors to contribute all their findings in the field to bridge the current knowledge gap and provide a stronger database for the coming years.

Tips and Tricks and Clinical Outcome of Cryopreserved Human Amniotic Membrane Application for the Management of Medication-Related Osteonecrosis of the Jaw (MRONJ): A Pilot Study.

Medication-related osteonecrosis of the jaw (MRONJ) is a complication of certain pharmacological treatments such as bisphosphonates, denosumab, and angiogenesis inhibitors. There are currently no guidelines on its management, particularly in advanced stages. The human amniotic membrane (hAM) has low immunogenicity and exerts anti-inflammatory, antifibrotic, antimicrobial, antiviral, and analgesic effects. It is a source of stem cells and growth factors promoting tissue regeneration. hAM acts as an anatomical barrier with suitable mechanical properties (permeability, stability, elasticity, flexibility, and resorbability) to prevent the proliferation of fibrous tissue and promote early neovascularization at the surgical site. In oral surgery, hAM stimulates healing and facilitates the proliferation and differentiation of epithelial cells in the oral mucosa and therefore its regeneration. We proposed using cryopreserved hAM to eight patients suffering from cancer (11 lesions) with stage 2-3 MRONJ on a compassionate use basis. A collagen sponge was added in some cases to facilitate hAM grafting. One or three hAMs were applied and one patient had a reapplication. Three patients had complete closure of the surgical site with proper epithelialization at 2 weeks, and two of them maintained it until the last follow-up. At 1 week after surgery, three patients had partial wound dehiscence with partial healing 3 months later and two patients had complete wound dehiscence. hAM reapplication led to complete healing. All patients remained asymptomatic with excellent immediate significant pain relief, no infections, and a truly positive impact on the patients' quality of life. No adverse events occurred. At 6 months of follow-up, 80% of lesions had complete or partial wound healing (30 and 50%, respectively), while 62.5% of patients were in stage 3. Radiological evaluations found that 85.7% of patients had stable bone lesions (n = 5) or new bone formation (n = 1). One patient had a worsening MRONJ but remained asymptomatic. One patient did not attend his follow-up radiological examination. For the first time, this prospective pilot study extensively illustrates both the handling and surgical application of hAM in MRONJ, its possible association with a collagen sponge scaffold, its outcome at the site, the application of multiple hAM patches at the same time, and its reapplication.

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.