Amniotic membrane (AM) and amnion/chorion foetal membranes (ACM) are mainly composed of collagen & laminin layers and constitute relatively new materials to the dental market. They have proven effective for periodontal treatments such as Guided Tissue Regeneration (GTR) [1–3]. Based on our expertise in the field of lyophilisation & securisation of human bone allograft (Phoenix® process), we aimed to develop our own process applied to ACM and to control its in vivoefficacy in GTR indication. Human placentas were donated under informed consent. ACM were separated from placenta and processed with a proprietary AMTRIX (TBF) Process. Resulting product was called ACMTRIX. The effectiveness of ACMTRIX in GTR was evaluated using an in vivorat calvaria defect model as followed:. Empty defect (2 animals),. ACMTRIX apposed onto the defect (4 animals),. 3 Bone substitutes (allogenic – mineralized cortical bone powder (Phoenix®); demineralized cancellous bone powder mixed with hydroxyapatite and demineralized bone matrix (DBM) cancellous block) filled in the defect and covered by ACMTRIX (4 animals). One animal per study group was sacrificed after 8 weeks, all others after 8 weeks. Evaluations were performed by: macroscopic observations, X Ray micro-CT, and histological analysis. For all groups using ACMTRIX, no major sign of inflammation were observed macroscopically and histologically. Moreover, bone tissue was already mature from 8 weeks and bone filling was slight to moderate. The higher mean rate of mineralization was obtained for the group associating DBM cancellous block + ACMTRIX. Although a xenogenic material, ACMTRIX was very well integrated without significant inflammatory reaction compared to empty defect and fully integrated in subcutaneous area. The mineralization was superior with DBM cancellous block probably thanks to the stabilization of the material in the defect. Used alone, ACMTRIX has no osteogenic potential. In conclusion, ACMTRIX has the potential to function as barrier for GTR and the unique properties associated with this material can augment its potential as a matrix for periodontal regeneration

Our musculoskeletal system has a limited capacity for repair. This has led to increased interest in the development of tissue engineering and biofabrication strategies for the regeneration of musculoskeletal tissues such as bone, ligament, tendon, meniscus and articular cartilage. This talk will demonstrate how different musculoskeletal tissues, specifically cartilage, bone and osteochondral defects, can be repaired using emerging biofabrication and 3D bioprinting strategies. This will include examples from our lab where cells and/or growth factors are bioprinted into constructs that can be implanted directly into the body, to approaches where biomimetic tissues are first engineered in vitro before in vivo implantation. The efficacy of these different biofabrication strategies in different preclinical studies will be reviewed, and lessons from the relative successes and failures of these approaches to tissue regeneration will be discussed.

Intervertebral disc (IVD) degeneration occurs with aging, leading to low back pain (LBP), which is one of the leading conditions of disability worldwide. With the lack of effective treatment, decellularized extracellular matrix (dECM) – based biomaterials have been proposed for IVD regeneration. However, the impact of donor ages on tissue repair had never been explored before in the disc field. Therefore, we aimed to address this question.

For that, a decellularization protocol for bovine nucleus pulposus (NP) of different aged donors (fetus, young and old) was optimized by testing several detergents (SDS and Triton). The process efficiency was evaluated in terms of DNA and cell removal, as well as ECM preservation. Afterwards, dECMs were repopulated with bovine NP cells and cultured ex vivo. At day 7, cell behavior, ECM de novo synthesis and remodeling were evaluated [1]. Moreover, dECMs’ inflammatory response was assessed after in vivo CAM assay. Finally, inflammatory and angiogenic cytokines were analyzed in the conditioned media-derived from dECMs by using a cytokine array.

As results, an optimal decellularization protocol (SDS 0.1%, 1h), efficient at removing cells and DNA from bovine NPs, while preserving ECM cues of native tissues, was developed. After repopulation, aggrecan increased in younger NPs, while collagen 2 decreased which may be indicative of matrix remodeling [1]. After in vivo CAM assay, fetal dECMs showed the highest inflammatory response. Finally, no statistically significant changes of cytokines were detected in the matrices, despite for a trend of higher IFN-α, IFN-γ and LIF in fetal dECMs, IL-1β in young dECMs and Decorin in old dECMs.

Overall, this work uncovered the importance of tissue donor ages for tissue regenerative purpose, opening new avenues for the development of appropriate therapeutic strategies for IVD degeneration.

Acknowledgments: FCT, EUROSPINE, ON Foundation.

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure.

Regenerative medicine (RM) promises to restore both the mechanical functionality and the biological composition of tissues after damage. Three-dimensional scaffolds are used in RM to host cells and let them produce proteins that are the building blocks of the native tissues. While regenerating tissues evolve over time through dynamic biomechanical and biochemical changes, current scaffolds’ generation are passive causing mechanical mismatch, suboptimal growth, and pain. Furthermore, current scaffolds ignore the complexity of the reciprocal bio-mechanics regulation, hindering the design of the next-gen scaffolds. To regenerate tissues and organs, biofabrication strategies that impart spatiotemporal control over cell-cell and cell-extracellular matrix communication, often through control over cell and material deposition and placement, are being developed. To achieve these targets, the spatiotemporal control over biological signals at the interface between cells and materials is often aimed for. Alternatively, biological activity can be triggered through the control of mechanical cues, harnessing more fundamental know-how in mechanobiology that could be combined with biofabrication strategies. Here, I present some of our most recent advancements in merging mechanobiology with biofabrication that enabled the control of cell activity, moving towards enhanced tissue regeneration as well as the possibility to create more complex 3D in vitro models to study biological processes.

Biofabrication is a popular technique to produce personalized constructs for tissue engineering. In this study we combined laponite (Lap), gellan gum (GG) with platelet-rich plasma (PRP) aiming to enhance the endothelial regeneration through the synergistic effects of their individual properties. Laponite has the ability to form porous three-dimensional networks mimicking the extracellular matrix structure, and PRP delivery of growth factors stimulates the endothelial cell proliferation and migration, offering a composite bioink for cell growth and support. The sustained release of these growth factors from the GG-laponite-PRP composite material over time provides a continuous source of stimulation for the cells, leading to more effective tissue engineering strategies for endothelial tissue regeneration. Four blend compositions comprising 1% w/v GG and 0.5 or 1% w/v Lap and 25% v/v PRP were combined with Wharton jelly mesenchymal stem cells (WJ-MSCs) and bioprinted into vessel-like structures with an inner diameter of 3 mm and a wall thickness of 1 mm. Stress/strain analysis revealed the elastomeric properties of the hydrogels with Young modulus values of 10 MPa. Increasing the Lap concentration led to a non-significant decrease of swelling ratio from 93 to 91%. Live/dead assay revealed cell viability of at least 76%, with the 0.5%Lap-GG viability exceeding 99% on day 21. Gradual increase of glycosaminoglycans accumulation and collagen production indicate promotion of ECM formation. The expression and membranous localization of PECAM-1 from day 7 and the granular intracellular localization of vWF after 2 weeks demonstrate in vitro endothelial functionality. In vivo subcutaneous implantation indicated the absence of any adverse immunological reactions. The results reveal the expression of both vWF and PECAM-1 by WJ-MSCs entrapped in all four construct compositions with significantly higher expression of vWF in the presence of PRP.

Introduction

The objective of the work is construction of a multi-bioactive scaffold based on that allows a space/time control over the regeneration of damaged bones by Medication-Related Osteonecrosis of the Jaw using a minimal invasive approach based on the injection of the fast-degrading pro neuro and angiogenic ELR (Elastin-Like Recombinamers) based hydrogels.

Abstract

The successful application of smart implantable devices requires materials used to easily adapt and respond to their microenvironment via physical and chemical cues. Nanotopography, a known important factor in cellular processes (i.e. cellular adhesion, proliferation, and, differentiation), has become a central approach to imparting clinically relevant materials with bioactive and biomimetic properties. This work focuses on the use of Directed irradiation synthesis (DIS), to create nanostructures on dissimilar materials including surfaces of metals, semiconductors, and polymers. DIS is a novel method that allows for the tuning of both surface nanoscale topography and surface chemistry through the tailoring of ion beam parameters, including energy and fluence. The application of DIS to direct cellular interactions on Ti6Al4V, MgAZ31, and PEEK is presented. Topography and chemistry changes at the nanoscale were characterized by SEM, XPS, AFM, and Contact Angle. In vitro tests were performed using macrophages (JJ741A) and human aortic and bone marrow mesenchymal stem cell (MSCs). DIS promotes an advanced cell adhesion state where cells are orientated following the designed nanofeatures in all irradiated specimens. A delay on immune response due to low levels of TNFa and higher levels of IL10 on irradiated Ti6Al4V were observed. Modified PEEK showed 3-fold higher ALP content at 7 days compared to pristine samples, and porous MgAZ31 treated with DIS revealed lower corrosion state and increased cell proliferation of HBMMSCs. Controlling the nanopatterning in biomaterials using DIS enables the design of bioactive surfaces to highly promote implant integration and tissue regeneration.

Introduction: The clinical need for a biodegradable material with broad application is evidenced by the fact that tissue loss as a result of injury or disease provides reduced quality of life for many at significant socio-economic cost. The development of simple biodegradable materials, with broad applicability and tissue/ cell specificity has to date proved elusive. Natural biopolymers such as alginate and chitosan are structural biomaterials of increasing significance to tissue repair and regeneration due to their potential for fabrication, design and efficient, environmentally benign synthesis. We describe the development of innovative microcapsule scaffolds based on chitosan and alginate that can be tailored to a range of cell types for a variety of tissues.

Methods: Semi-permeable polysaccharide microcapsules were produced by a one-step method, in which the deposition of a semi-permeable alginate/chitosan membrane around droplets of sodium alginate was coupled with in-situ precipitation of amorphous calcium phosphate as described by Leveque et al (2002)*. A variety of human cell types including mesenchymal stem cells, osteoprogenitors selected using the STRO-1 antibody by magnetically activated cell separation (MACS), osteoprogenitors transfected with adenovirus expressing Green Fluorescent Protein (GFP) and chondrocytes were mixed with sodium alginate and encapsulated within alginate/chitosan and calcium phosphate.

Results: Hybrid spheres (750–10,000um) were generated encapsulating primary human osteoprogenitor cells, STRO-1 selected osteoprogenitors and AdGFP transfected osteoprogenitors. Encapsulated cells remain viable inside the polysaccharide microcapsules for 2 weeks as shown by positive alkaline phosphatase staining of encapsulated cells. Cells expressing GFP were observed within microspheres indicating the e ability to deliver cells/factors as well as the potential for gene therapy. Encapsulation and delivery of active BMP-2 was confirmed using the promyoblast cell line C2C12 known to be exquisitely sensitive to BMP-2. Nucleation of calcium phosphate occurred within the polysaccharide membrane and could be controlled by the phosphate concentration in the alginate droplets to produce hybrid microcapsules with enhanced mechanical strength. Thin walled capsules were shown to split and degrade in culture within 2–4 days releasing viable osteoprogenitor cells indicating the ability to manipulate the mechanical integrity and to programme degradation of the microspheres. Finally we have shown that aggregation of the microspheres into extended frameworks can be achieved using a designed droplet/vapour aerosol system resulting in foams of aggregated beads.

Discussion and Conclusion: A variety of human skeletal cells have been encapsulated within polysaccharide/ calcium phosphate microspheres and extended frameworks with specifiable dimensions. These composite scaffolds offer stable mechanical and chemical biomimetic environments conducive to normal cell function. Natural polysaccharides are also highly amenable to complexation with a range of bioactive molecules and consequently offer tremendous potential in tissue engineering and regeneration of hard and soft tissues.

Cell-based scaffold-free tissue equivalents present a limited clinical translation as consequence of the delayed extracellular matrix (ECM) deposition due to the prolonged production time in vitro. Different combinations of media supplements such as ascorbic acid, growth factors, oxygen tension among others can modulate the cell fate or the ECM synthesis. New research lines are focusing on the use of macromolecular crowders (MMCs) as media supplement for cell sheet production due to their ability to increase ECM deposition by volume exclusion effect, pro-collagenases alosteric regulation, matrix self-assembly by confinement and diffusion limitation (most probably, modulating the interaction between the ECM, MMPs and TIMPs). Herein, different molecular weights and concentrations of a natural potential MMC (Crowder-A) have been tested in equine adipose-derived stem cell (eADSC) and human dermal fibroblast (hADF) cultures in comparison with other commonly used crowders such as carrageenan and the Ficoll™ cocktail 70 KDa and 400 KDa. The eADSCs were characterized according to the current criteria for horse MSCs. Tri-lineage and FACS analysis showed eADSC osteogenic and adipogenic potentials and the presence of the markers CD29, CD44, CD90. The screening of the aforementioned Crowder-A was performed in cultures of 15,000 cells / cm² for the eADSCs and 25,000 cells / cm² for the hADFs during 3, 5, and 7 days. Non-MMC conditions were used as negative controls. Collagen type I was analysed by SDS-PAGE. Other collagen types were studied by immunocytochemistry assays. Significant increase of some ECM components was observed in some concentrations and molecular weights of the Crowder-A.

Research in orthopaedics is now moving away from permanent metallic implants, and looking towards the use of bioresorbable polymers (e.g. PLLA, PGA and related co-polymers) that, when implanted into the injured site, bioresorb as the tissue heals. However, reports of a delayed inflammatory reponse occurring in the late stages of polymer degradation has limited the wide scale use of these polymers. Few studies assess the long-term biocompatibility of these polymers and with an increasing market for bioresorbable materials it is anticipated that this will be a future issue. This work aims to develop a predictive tool that can be used to assess the delayed inflammatory response of poly(D,L-lactide-co-glycolide) (PDLGA) using in vitro tests. An elevated temperature accelerated test (47^oC) was developed and utilitised to induce predetermined amounts of degradation in PDLGA. This was used to mimic a range of clinically relevant in vivo implantation times up to 5–6 months. All pre-degradion work was performed under sterile conditions, in PBS solution. At predetermined time intervals, indicators of late stage inflammation will be assessed using an MTT cytotoxicity assay, an inflammation antibody array and an ELISA analysis for inflammatory factors, with mouse L929 fibroblasts, RAW264.7 and primary BMDM macrophages. It is hypothosised that at the later degradation time intervals signs of inflammatory factors will be observed. The methodologies developed in this work can be applied to the optimisation of polymer degradation profiles to minimise late-stage inflammatory repsonse and identification of beneficial additives in this regard.

Purpose: To assess the use of abdominal aorta cryopreserved allografts as guided regeneration membranes in long bone defects.

Materials and methods: This is a prospective randomized blind study of 10 White New Zealand rabbits. 10 mm-long diaphyseal defects were created in both radii: on one side the defect was separated from the surrounding tissue by means of a tube-shaped cryopreserved aortic allograft; the contralateral radius (control) was left to develop spontaneously with no membrane. The animals were put down after 6, 12, 24 and 30 months. A whole range of different studies were made: x-rays, CT, MRI, morphodensitometric techniques and optical and electronic microscopy.

Results: No complete bone regeneration was observed in any of the controls. In 9 out of the 10 defects for which an aortic allograft was used complete bone regeneration was achieved as well as a restoration of continuity with a corticomedullary pattern. A progressive increase in density and thickness was observed in the regenerated cortex, which reached values similar to those of normal bone. A gradual reduction of the medullary/cortical thickness index was also detected.

Discussion: The microscopic images taken suggest that cryopreserved arterial allografts used in guided regeneration behave like barrier membranes and as osteoinductive agents because of the osteoblastic differenciation of endothelial and/or muscular cells and/or ossification secondary to proteic changes in the extracellular matrix of the artery. This could be regarded as the application of artery calcification and ossification (usually associated with arteriosclerosis, ageing, diabetes and renal failure) to the regeneration of bone defects.

Conclusions: It is possible to use cryopreserved aortic allografts as osteostimulating membranes in the guided regeneration of bone defects.

Purpose

To assess performance of a polyurethane scaffold designed to facilitate regrowth of tissue after irreparable partial meniscus tissue loss.

Purpose: Regeneration of skeletal tissue for fracture repair or during morphogenesis involves common phases of cell proliferation and differentiation. Mesenchymatous precursor cells have multiple origins. These cells can be identified in the bone marrow, in the deep layer of the periosteum and in the endosteum. More recently, the presence of circulating multipotent stem cells has been demonstrated in the general circulation. Their contribution to skeletal regeneration processes is suspected. The experiments we report allow visualisation of the multidirectional differentiation phenomena involving mesenchymatous precursor cells in an animal model of skeletal tissue regeneration.

Material and methods: An experimental surgical protocol was developed to study the regeneration of skeletal tissue in New Zealand rabbits. Eighteen animals were used. A vascularised periosteum flap was transferred onto the medial aspect of the knee. The flap was fixed in order to be exposed to flexion and extension stress during spontaneous ambulation. The joint was not damaged in any way and the adjacent bone segments were left intact. The animal was allowed to move freely postoperatively. The animal was sacrificed two days to eight weeks later to study standard histological slices taken from the regenerate region and the recipient knee joint.

Results: The zone of influence of the flap was recognised early in the environment where it was apposed. This zone involved the marrow of the metaphyseal regions, the neighbouring muscles, the joint cavity, and the menisci. Cell proliferation was noted in each of these sites. It was associated with differentiation of the precursor elements in multiple directions of the mesenchymatous lines. This led to production of cartilaginous, bony, fibrous, and even muscle tissue in the medullary cavity, in the menisci, and in the open joint space. Immunohistochemistry demonstrated the contribution of the mesenchymatous stem cells whose circulating pool was visualised.

Discussion: This work is in agreement with the recent demonstration of the contribution of stem cells to general healing phenomena, and the physiological turnover of healthy tissue.

Conclusion: The strong potential of multipotent stem cells for tissue reparation and regeneration processes opens important perspectives for cell therapy and tissue engineering. The demonstration of physiological processes operating in vivo which involve participation of the endogenous cell pool is of importance for all fields of medicine and surgery for the treatment of the musculoskeletal system.

Introduction: Partial meniscectomy is the preferred treatment option for patients with irreparable meniscal tears. While generally accepted as producing favorable clinical results in the short run, it is widely accepted that meniscectomy induces articular cartilage degeneration in the long run. Thus, tissue regeneration post-meniscectomy is a desirable therapeutic approach in order to restore the function of the meniscus, thereby preventing long-term damage. A novel device, designed to act as a scaffold for blood vessel ingrowth and meniscal tissue regeneration in patients with irreparable meniscus tears and meniscal tissue loss, has recently been developed.

Methods: Fifty-two patients with an irreparable medial or lateral meniscal tear or partial meniscus loss, with intact rim, were treated with the meniscal scaffold in this prospective, non-randomised, single-arm, multi-centre clinical study. To date, biopsy samples 12 months post-implantation harvested from the center of the inner rim of the implanted scaffold meniscus using a standardized biopsy harvest protocol are available from 9 of the 52 patients. Histochemical staining was performed with haematoxylin and eosin, Masson’s trichrome, Sirius Red and combined Periodic Acid Schiff-Alcian Blue (PAS-AB). Immunohistochemistry was performed using the cartilage markers S100, the vessel markers CD31 and CD34, the smooth muscle marker SMA, and the histiocytic marker CD68.

Results: All biopsies showed fully vital material, with no signs of necrosis or cell death. In addition to a fibrous capsule, 3 distinct zones were identified based on the presence or absence of vessel structures, cellular morphology, and composition of extracellular matrix. Zone 1, a vascularized, fibrotic zone, mainly consisting of fibroblasts, was observed in 4/9 biopsies. Zone 2, an avascular and loose collagenized zone, consisting of a mixture of fibroblasts and chondrofibroblast-like cells, and Zone 3, an avascular and fibrin-rich zone, consisting of fibrochondroblast-like cells, were evident in all 9 biopsies.

Conclusions: All biopsies showed complete re-population, and thus can be regarded as vital structures, illustrating the biocompatibility of the meniscal scaffold. Moreover, zonal organisation, each with its own histological characteristics, suggests an ongoing process of regeneration, maturation and integration towards meniscus-like tissue. These data offer a first insight into the complex human healing potential after implantation of a polyurethane meniscus scaffold.

On behalf of the Actifit Study Group: R Verdonk, P Beaufils, J Bellemans, P Colombet, R Cugat, P Djian, H Laprell, P Neyret, H Paessler,

In the last decades, significant effort has been attempted to salvage the meniscus following injury. Basic science approaches to meniscus repair include procedures for both meniscus regeneration and meniscus healing. Regeneration of meniscal tissue focuses on filling a defect with reparative tissue, which resembles the native structure and function of the meniscus. Procedures for meniscus healing, on the other hand, aim to accomplish adhesion between the margins of a meniscal lesion, with no attempt to regenerate or replace meniscal tissue. Regeneration studies of tissue to fill a defect in the meniscus have shown interesting results, but complete restoration of the native meniscus has not yet been accomplished. Healing of a meniscal lesion has been investigated in different models although none has demonstrated reproducible healing. Therefore, different paths of investigation must be undertaken, and one of these may be the cell-therapy / tissue engineering approach. In a study from our group, we showed the capacity of chondrocyte-seeded cartilaginous scaffold to repair a bucket-handle lesion of the knee meniscus orthotopically in a large animal study. Following studies were done in order to test the potential of other scaffolds and different cell sources for the repair of the meniscal tissue. We have also evaluated the role of hypoxia in meniscal development in vitro as basis for future research in this field, as hypoxia could be be considered as a promoter for meniscal cells maturation, and opens considerably opportunities in the field of meniscus tissue engineering

Purpose: The aim of our study was to evaluate bone formation and angiogenesis produced within a biodegradable poly-D, L-lactide-co-glycolide acid/calcium phosphate (PLGA/CaP) scaffold when used to treat a diaphyseal tibia defect and compare this to an iliac crest autograft or an empty defect. Method: An 8.0 mm diaphyseal defect was created in a canine tibia model. All tibiae were reamed to 7.0 mm and fixed with a 6.5 mm statically locked intramedullary nail. Eighteen canines were allotted into three treatment groups:. empty (N=5),. iliac crest autograft (N=6), or. PLGA/CaP biodegradable scaffold Tissue Regeneration Therapeutics Inc., ON, Canada) (N=7). Fluorescent markers were given at different times: calcein green (six weeks), xylenol orange (nine weeks), and tetracycline (11 and 14 weeks). Animals were sacrificed at 15 weeks and perfused with a barium compound. Radiography, Micro CT, and brightfield and fluorescent microscopy were used for analysis. Results: Micro CT and brightfield images of scaffold samples displayed multiple vessels (10 to 100μm) within the scaffold. The bone volume and vasculature volume (measured with Micro CT) within the tibial defect site were reported as a percentage of the total volume of the defect site. The percent bone volume within the defect site was not different between treatment groups (p=0.112). There was greater percent vasculature volume in the scaffold group than the autograft group (p< 0.001). Bone formation at the osteotomy sites was defined as the distance from the original osteotomy site to the tip of newly formed bone. Osteotomy bone formation was significantly greater in the scaffold group than the autograft group (p=0.015). Osteotomy sites associated with greater angiogenesis displayed greater bone formation. Bone formation rates were reported as the distance between the fluorescent bone labels. Autograft samples had the greatest bone formation rates within the periosteum. Autograft and scaffold samples had the greatest rate of bone formation within the cortex. Conclusion: Our canine tibial defect model provides a satisfactory facsimile of the traumatic tibia fracture with associated bone loss. The PLGA/CaP biodegradable scaffold we have employed promotes angiogenesis within a defect and could be used in conjunction with autografting