Quality by Design: Development of Safe and Efficacious Full-Thickness Acellular Dermal Matrix Based on EuroGTPII Methodologies.

Background: The activities of tissue establishments are constantly and rapidly evolving. The development of a new type of allograft, full-thickness acellular dermal matrix, with high mechanical properties to be used in tendon repair surgeries and abdominal wall reconstruction, has determined the need for quality by design process in order to assess evidence of quality, safety and efficacy. The EuroGTPII methodologies were specifically tailored to perform the risk assessment, identify and suggest tests in order to mitigate the potential risk consequences of a novel tissue preparation implementation. Methods: The new allograft and associated preparation processes were assessed using the EuroGTP methodologies and characterized to properly evaluate the novelty (Step 1), identify and quantify the potential risks and risk consequences (Step 2), and define the extent of pre-clinical and clinical assessments required to mitigate the risks identified in the assessment (Step 3). Results: Four risk consequences associated with the preparation process were identified: (i) implant failure related with tissue procurement and the reagents used during the decellularization protocol; (ii) unwanted immunogenicity related with the processing; (iii) disease transmission linked with the processing, reagents used, reduction in the reliability of microbiology testing and the storage conditions; and (iv) toxicity related to the reagents used and handling of the tissue during clinical application. The outcome of the risk assessment was a low level of risk. Nevertheless, it determined the need for a series of risk mitigation strategies proposed to reduce each individual risk and to provide additional evidence of the safety and efficacy of full-thickness acellular dermal matrix grafts. Conclusion: EuroGTPII methodologies allow us to identify the risks and ensure the correct definition of pre-clinical assessments required to address and mitigate the potential risk consequences, before proceeding with clinical use of the new allografts in patients.

Decellularized Graft for Repairing Severe Peripheral Nerve Injuries in Sheep.

BACKGROUND AND OBJECTIVES: Peripheral nerve injuries resulting in a nerve defect require surgical repair. The gold standard of autograft (AG) has several limitations, and therefore, new alternatives must be developed. The main objective of this study was to assess nerve regeneration through a long gap nerve injury (50 mm) in the peroneal nerve of sheep with a decellularized nerve allograft (DCA). METHODS: A 5-cm long nerve gap was made in the peroneal nerve of sheep and repaired using an AG or using a DCA. Functional tests were performed once a month and electrophysiology and echography evaluations at 6.5 and 9 months postsurgery. Nerve grafts were harvested at 9 months for immunohistochemical and morphological analyses. RESULTS: The decellularization protocol completely eliminated the cells while preserving the extracellular matrix of the nerve. No significant differences were observed in functional tests of locomotion and pain response. Reinnervation of the tibialis anterior muscles occurred in all animals, with some delay in the DCA group compared with the AG group. Histology showed a preserved fascicular structure in both AG and DCA; however, the number of axons distal to the nerve graft was higher in AG than in DCA. CONCLUSION: The decellularized graft assayed supported effective axonal regeneration when used to repair a 5-cm long gap in the sheep. As expected, a delay in functional recovery was observed compared with the AG because of the lack of Schwann cells.

Comparison of a human acellular dermal matrix and a polypropylene mesh for pelvic floor reconstruction: a randomized trial study in a rabbit model.

Non-absorbable polypropylene (PP) meshes have been widely used in surgical reconstruction of the pelvic floor disorders. However, they are associated with serious complications. Human acellular dermal matrices (hADM) have demonstrated safety and efficacy in reconstructive medicine, but their suitability and efficacy at vaginal level is not known. This study compares the biological performance of PP mesh and a newly developed hADM. 20 rabbits were randomized to receive the hADM graft or the PP mesh. Grafts were surgically implanted in the abdominal wall and vagina. After 180 days, grafts were explanted and evaluated. The vaginal mesh extrusion rate was higher in the PP group (33% vs. 0%, p = 0.015). Full integration of the vaginal grafts was more frequent in the hADM group, where 35% of the grafts were difficult to recognize. In the PP group, the vaginal mesh was identified in 100% of the animals (p = 0.014). In PP group, the infiltrates had a focal distribution and were mostly located in the internal part of the epithelium, while in the hADM group, the infiltrates had a diffuse distribution. Additionally, the hADM group also presented more B-lymphocytes and less T-lymphocytes. Biomechanical analysis showed that hADM had lower resistance to stress. Moreover, PP mesh stiffness and elasticity were higher. Then, hADM is associated with fewer clinical complications, as well as better tissue integration. However, it shows greater incorporation into the surrounding native tissue, especially in the vaginal location, undergoing a reduction in its biomechanical properties 6 months after implantation.

A Comparative Study Using Fluorescent Confocal Microscopy and Flow Cytometry to Evaluate Chondrocyte Viability in Human Osteochondral Allografts.

The preservation conditions of fresh osteochondral allografts for clinical applications are critical due their objective: to transplant mature hyaline cartilage containing viable chondrocytes, maintaining their metabolic activity and also preserving the structural and functional characteristics of the extracellular matrix. The aim of the present study was to compare fluorescence confocal microscopy and flow cytometry techniques to evaluate the viability of the chondrocytes present in the osteochondral tissue, in order to determine their effectiveness and thus ensure reproducibility and robustness of the analysis. To this end, osteochondral allografts from human cadaveric donors were preserved at 4 °C for 3 weeks in a preservation medium supplemented with antibiotic and antifungal agents. Cell viability of chondrocytes was determined by monitoring the cartilage for 3 weeks of preservation by confocal fluorescence microscopy and flow cytometry, obtaining cell viabilities of 83.7 ± 2.6% and 55.8 ± 7.8% for week three, respectively. The confocal fluorescence microscopy approach is more advantageous and accurate, as it correlates better with actual cell viability values for monitoring osteochondral graft preservation, detecting only the cells that died a natural death associated with the preservation method.

A novel decellularized nerve graft for repairing peripheral nerve long gap injury in the rat.

Decellularized nerve allografts are an alternative to autograft for repairing severe nerve injuries, since they have higher availability and do not induce rejection. In this study, we have assessed the regenerative potential of a novel decellularization protocol for human and rat nerves for repairing nerve resections, compared to the gold standard autograft. A 15-mm gap in the sciatic nerve was repaired with decellularized rat allograft (DC-RA), decellularized human xenograft (DC-HX), or fresh autograft (AG). Electrophysiology tests were performed monthly to evaluate muscle reinnervation, whereas histological and immunohistochemical analyses of the grafts were evaluated at 4 months. A short-term study was also performed to compare the differences between the two decellularized grafts (DC-RA and DC-HX) in early phases of regeneration. The decellularization process eliminated cellularity while preserving the ECM and endoneurial tubules of both rat and human nerves. Higher amount of reinnervation was observed in the AG group compared to the DC-RA group, while only half of the animals of the DC-HX showed distal muscle reinnervation. The number of regenerating myelinated axons in the mid-graft was similar between AG and DC-RA and lower in DC-HX graft, but significantly lower in both DC grafts distally. At short term, fibroblasts repopulated the DC-RA graft, supporting regenerated axons, whereas an important fibrotic reaction was observed around DC-HX grafts. In conclusion, the decellularized allograft sustained regeneration through a long gap in the rat although at a slower rate compared to the ideal autograft, whereas regeneration was limited or even failed when using a decellularized xenograft.

"Off-the-Shelf" Nerve Matrix Preservation.

Background: Decellularized human nerves overcome the limitations of the current treatments for large peripheral nerve injuries. However, the use of decellularized nerves requires an "off-the-shelf" availability for useful and actual clinical application. In this study, we addressed the preservation of the native and decellularized human nerve matrix in an integrative approach for tissue scaffold production. Materials and Methods: For native nerve matrix preservation analysis, we used histological examination and immunofluorescence to examine the structure, biomechanical assays to evaluate the tensile strength and Young's modulus, and analyzed the extracellular matrix (ECM) composition using enzyme-linked immunosorbent assay (ELISA) and biochemical assays for laminin, collagen and sulfated glycosaminoglycans (sGAG). After decellularization, nuclear remnants and DNA content were evaluated using 4',6-diamidino-2-phenylindole (DAPI) staining and the picogreen quantification assay, as well as immunofluorescence or ELISA for cell rests (S100 protein and myelin staining) evaluation. Decellularized cryopreserved scaffolds were assayed for biomechanics, ECM composition, and structural maintenance. Cytotoxicity assays were performed to evaluate the biocompatibility of the nerve matrix extracts after cryopreservation. Results: We compared different strategies for native nerve storage and found that preservation up to 7 days at 4°C in Roswell Park Memorial Institute medium maintained biomechanical properties, such as Young's modulus and tensile strength, along with the structure and ECM composition, regarding laminin, collagen, and sGAG. After a successful decellularization process, that eliminated cell remnants, nerve scaffolds were frozen in an "in house" formulated cryoprotectant, using an automatic controlled rate freezer. Nerve structure, ECM composition, and biomechanical properties were maintained before and after the freezing process in comparison with native nerves. The extracts of the nerve scaffolds after thawing were not cytotoxic and the freezing process sustained good viability in 3T3 cells (graphical abstract). Conclusion: Since our approach facilitates transport, storage, and provide a ready-to-use alternative, it could be used in a clinical application for the treatment of long-gap peripheral nerve injuries in regenerative medicine.

Rabbit as an animal model for the study of biological grafts in pelvic floor dysfunctions.

The aims of this study were to evaluate the feasibility of the New Zealand White (NZW) rabbit for studying implanted biomaterials in pelvic reconstructive surgery; and to compare the occurrence of graft-related complications of a commercial polypropylene (PP) mesh and new developed human dermal matrix implanted at vaginal and abdominal level. 20 white female NZW rabbits were randomized into two groups, experimental group (human acellular dermal matrices-hADM-graft) and control group (commercial PP graft). In each animal, grafts were surgically implanted subcutaneously in the abdominal wall and in the vaginal submucosa layer for 180 days. The graft segments were then removed and the surgical and clinical results were analyzed. The main surgical challenges during graft implantation were: (a) an adequate vaginal exposure while maintaining the integrity of the vaginal mucosa layer; (b) to keep aseptic conditions; (c) to locate and dissect the breast vein abdominal surgery; and (d) to withdraw blood samples from the ear artery. The most abnormal findings during the explant surgery were found in the PP group (33% of vaginal mesh extrusion) in comparison with the hADM group (0% of vaginal graft extrusion), p = 0.015. Interestingly, macroscopic observation showed that the integration of the vaginal grafts was more common in the hADM group (40%) than in the PP group, in which the vaginal mesh was identified in 100% of the animals (p = 0.014). The NZW rabbit is a good model for assessing materials to be used as grafts for pelvic reconstructive surgery and vaginal surgery. Animals are easily managed during the procedures, including surgical intervention and vaginal mucosa approach. Additionally, hADM is associated with fewer clinical complications, as well as better macroscopic tissue integration, compared to PP mesh.

Which type of bone releases the most vancomycin? Comparison of spongious bone, cortical powder and cortico-spongious bone.

Bone infections can be challenging to treat and can lead to several surgeries and relapses. When a graft is needed, cavitary bone loss can be grafted with cancellous or cortical bone. Both can be used for grafting. However, the antibiotic releasing capacity of these grafts has not been compared. Which type of bone is best at releasing the most antibiotic has not been well established. The aim of this study was to determine which type of bone is best for antibiotic release when the bone is suffused with antibiotics by the surgeon. The hypothesis is that there would be a difference between the type of bone tested due to different release capacities of cortical and cancellous bone. This was an experimental study. Cortical spongy bone in chips, Spongy bone in chips and demineralized cortical bone powder were compared. For each type of bone, 5 samples were tested. Processed and decontaminated grafts were freeze-dried to be kept at room temperature. The primary endpoint was the amount of vancomycin released by the graft as it affects the concentration of antibiotic around the graft in clinical practice. The procedure for the study consisted of full graft immersion in a vancomycin solution. Then, the liquid was removed with aspiration. In order to measure the quantity of antibiotic released, the bone was put into distilled water in agitation in a heated rocker at 37 °C. After 30 min of soaking, 1 mL of the liquid was removed. The same extraction process was also carried out after 60 min soaking, 2 h, 3 h, 24 h, and 48 h. No differences were found between each type of bone relative to the concentration of vancomycin released at each time of the assessment. There was a significant difference in the weight of the bone with a higher weight for the cortical powder (1.793 g) versus cortical spongy bone and spongy bone (1.154 g and 1.013 g) with a p value < 0.0001. A significant difference was seen in the weight of the bone with vancomycin after the aspiration of the liquid with 3.026 g for cortical powder, 2.140 g and 2.049 g for the cortical spongy bone and the spongy bone with a p value < 0.0001. In daily clinical practice, one can use cancellous bone, cortico-cancellous bone or cortical powder in order to add vancomycin to a bone graft. Our results show the release kinetics of the soaked allografts. With a maximum of 14 mg/mL in the first minutes and a rapid decrease it shows a pattern comparable to antibiotic loaded bone cement. The method used appears favourable for prophylactic use, protecting the graft against contamination at implantation, but is not sufficient for treating chronic bone infection. LEVEL OF EVIDENCE: V.

Effective and durable genetic modification of human mesenchymal stem cells via controlled release of rAAV vectors from self-assembling peptide hydrogels with a maintained differentiation potency.

Controlling the release of recombinant adeno-associated virus (rAAV) vectors from biocompatible materials is a novel, attractive approach to increase the residence time and effectiveness of a gene carrier at a defined target site. Self-assembling peptides have an ability to form stable hydrogels and encapsulate cells upon exposure to physiological pH and ionic strength. Here, we examined the capacity of the peptide hydrogel RAD16-I in a pure (RAD) form or combined with hyaluronic acid (RAD-HA) to release rAAV vectors as a means to genetically modify primary human bone marrow-derived mesenchymal stem cells (hMSCs), a potent source of cells for regenerative medicine. Specifically, we demonstrate the ability of the systems to efficiently encapsulate and release rAAV vectors in a sustained, controlled manner for the effective transduction of hMSCs (up to 80%) without deleterious effects on cell viability (up to 100%) or on their potential for chondrogenic differentiation over time (up to 21days). The present study demonstrates that RAD16-I is an advantageous material with tunable properties to control the release of rAAV vectors as a promising tool to develop new, improved therapeutic approaches for tissue engineering in vivo.

Bimolecular based heparin and self-assembling hydrogel for tissue engineering applications.

One major goal of tissue engineering is to develop new biomaterials that are similar structurally and functionally to the extracellular matrix (ECM) to mimic natural cell environments. Recently, different types of biomaterials have been developed for tissue engineering applications. Among them, self-assembling peptides are attractive candidates to create artificial cellular niches, because their nanoscale network and biomechanical properties are similar to those of the natural ECM. Here, we describe the development of a new biomaterial for tissue engineering composed by a simple combination of the self-assembling peptide RAD16-I and heparin sodium salt. As a consequence of the presence of heparin moieties the material acquired enhances the capacity of specific binding and release of growth factors (GFs) with heparin binding affinity such as VEGF165. Promising results were obtained in the vascular tissue engineering area, where the new composite material supported the development of tubular-like structures within a three dimensional (3D) culture model. Moreover, the new scaffold enhances the cell survival and chondrogenic commitment of adipose-derived stem cells (ADSC). Interestingly, the expression of specific markers of mature cartilage tissue including collagen type II was confirmed by western blot and real-time PCR. Furthermore, positive staining for proteoglycans (PGs) indicated the synthesis of cartilage tissue ECM components. Finally, the constructs did not mineralize and exhibited mechanical properties of a tissue undergoing chondrogenesis. Altogether, these results suggest that the new composite is a promising "easy to prepare" material for different reparative and regenerative applications.

Multivalent dendrimers presenting spatially controlled clusters of binding epitopes in thermoresponsive hyaluronan hydrogels.

The controlled presentation of biofunctionality is of key importance for hydrogel applications in cell-based regenerative medicine. Here, a versatile approach was demonstrated to present clustered binding epitopes in an injectable, thermoresponsive hydrogel. Well-defined multivalent dendrimers bearing four integrin binding sequences and an azido moiety were covalently grafted to propargylamine-derived hyaluronic acid (Hyal-pa) using copper-catalyzed alkyne-azide cycloaddition (CuAAC), and then combined with pN-modified hyaluronan (Hyal-pN). The dendrimers were prepared by synthesizing a bifunctional diethylenetriamine pentaacetic acid core with azido and NHBoc oligo(ethylene glycol) aminoethyl branches, then further conjugated with solid-phase synthesized RGDS and DGRS peptides. Azido terminated pN was synthesized by reversible addition-fragmentation chain transfer polymerization and reacted to Hyal-pa via CuAAC. Nuclear magnetic resonance (NMR), high performance liquid chromatography, size exclusion chromatography and mass spectroscopy proved that the dendrimers had well-defined size and were disubstituted. NMR and atomic absorption analysis confirmed the hyaluronan was affixed with dendrimers or pN. Rheological measurements demonstrated that dendrimers do not influence the elastic or viscous moduli of thermoresponsive hyaluronan compositions at a relevant biological concentration. Finally, human mesenchymal stromal cells were encapsulated in the biomaterial and cultured for 21days, demonstrating the faculty of this dendrimer-modified hydrogel as a molecular toolbox for tailoring the biofunctionality of thermoresponsive hyaluronan carriers for biomedical applications.

On the mechanism of Candida spp. photoinactivation by hypericin.

The photoprocesses involved in hypericin photoinactivation of three different Candida species (C. albicans, C. parapsilosis and C. krusei) have been examined. Production of singlet oxygen from the triplet state and of superoxide from both the triplet state and the semiquinone radical anion are demonstrated. Hydrogen peroxide is formed downstream of these early events. The outcome of the photodynamic treatments is dictated by the intracellular distribution of hypericin, which is different in the three species and affects the ability of hypericin to produce the different reactive oxygen species and trigger cell-death pathways. The results are in line with the previously-observed different susceptibilities of the three Candida species to hypericin photodynamic treatments.

In vitro fungicidal photodynamic effect of hypericin on Candida species.

Hypericin is a natural photosensitizer considered for the new generation of photodynamic therapy (PDT) drugs. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin PDT on various Candida spp., assessing its photocytotoxicity to keratinocytes (HaCaT) and dermal fibroblasts (hNDF) to determine possible side effects. A 3 log fungicidal effect was observed at 0.5 McFarland for two Candida albicans strains, Candida parapsilosis and Candida krusei with hypericin concentrations of 0.625, 1.25, 2.5 and 40 µm, respectively, at a fluence of 18 J cm(-2) (LED lamp emitting at 602 ± 10 nm). To obtain a 6 log reduction, significantly higher hypericin concentrations and light doses were needed (C. albicans 5 µM, C. parapsilosis 320 µM and C. krusei 320 µM; light dose 37 J cm(-2)). Keratinocytes and fibroblasts can be preserved by keeping the hypericin concentration below 1 µm and the light dose below 37 J cm(-2). C. albicans appears to be suitable for treatment with hypericin PDT without significant damage to cutaneous cells.