Fluorescent Reporter Mice for Nerve Guidance Conduit Assessment: A High-Throughput in vivo Model.

OBJECTIVE: Use of cell culture and conventional in vivo mammalian models to assess nerve regeneration across guidance conduits is resource-intensive. Herein we describe a high-throughput platform utilizing transgenic mice for stain-free axon visualization paired with rapid cryosection techniques for low-cost screening of novel bioengineered nerve guidance conduit performance. METHODS: Interposition repair of sciatic nerve transection in mice expressing yellow fluorescent protein in peripheral neurons (Thy1.2 YFP-16) was performed with various bioengineered neural conduit compositions using a rapid sutureless entubulation technique under isoflurane anesthesia. Axonal ingrowth was assessed at 3 and 6 weeks using epifluorescent microscopy following cryosectioning. RESULTS: Mean procedure time (incision-to-closure) was less than 2½ minutes. Direct operational costs of a 3-week experiment was calculated at $21.47 per animal. Tissue processing steps were minimized to aldehyde fixation, cryoprotection and sectioning, and rapid fluorescent dye staining for conduit visualization. Fluorescent microscopy readily resolved robust axonal sprouting at 3 weeks, with clear elucidation of ingrowth-permissive, semipermissive, or restrictive nerve guidance conduit environments. CONCLUSION: A rapid and cost-efficient in vivo platform for screening of nerve guidance conduit performance has been described. LEVEL OF EVIDENCE: NA. Laryngoscope, E392-E392, 2018.

A cost-effective cell- and matrix-based minimally invasive single-stage chondroregenerative technique developed with validated vertical translation methodology.

Introduction The morbidity and significant health economic impact associated with the chondral lesion has led to a large number of strategies for therapeutic neochondrogenesis. The challenge has been to develop techniques that are cost effective single-stage procedures with minimal surgical trauma that have undergone rigorous preclinical scrutiny and robust reproducible assessment of effectiveness. A biological repair requires the generation of a cellular and matrix composite with appropriate signalling for chondrogenic differentiation. Methods and results A technique was developed that allowed chondrogenic primary (uncultured) cells from bone marrow aspirate concentrate, combined with a composite hydrophilic and fibrillar matrix to be applied arthroscopically to a site of a chondral lesion. The construct was tested in vitro and in animal experiments before clinical trials. Clinical trials involved 60 patients in a prospective study. Symptomatic International Cartilage Repair Society grade 3 and 4a lesions were mapped and treated. Pre- and postoperative clinical assessments showed statistically significant improved outcomes; Lysholm Knee Scoring Scale (mean 52.8 to > 76.4; P < 0.05) International Knee Documentation Committee (mean 39 to > 79 P < 0.05) and Knee injury and Osteoarthritis Outcome Score (64.5 to >89.2 P < 0.05). Postoperative magnetic resonance imaging was evaluated morphologically (magnetic resonance observation of cartilage repair tissue, average MOCART score 72) and qualitatively; the regenerate was comparable to native cartilage. Conclusions This technique is effective, affordable, requires no complex tools and delivers a single-stage treatment that is potentially accessible to any centre capable of performing arthroscopic surgery. Good clinical results were found to be sustained at five years of follow-up with a regenerate that appears hyaline like using multiple magnetic resonance measures.

In vivo assessment of a nanofibrous silk tube as nerve guide for sciatic nerve regeneration.

A nanofibrous silk nerve conduit has been evaluated for its efficiency based on the promotion of peripheral nerve regeneration in rats. The designed tubes with or without Schwann cells were implanted into a 10 mm gap in the sciatic nerves of the rats. Four months after the surgery, the regenerated nerves were monitored and evaluated by macroscopic assessments and histology. The results demonstrated that the nanofibrous grafts, especially in the presence of Schwann cells, enabled reconstruction of the rat sciatic nerve trunk with a restoration of nerve continuity and formation of nerve fibres with myelination. Histological data demonstrated the presence of Schwann and glial cells in regenerated nerves. This study strongly supports the feasibility of using artificial nerve grafts for peripheral nerve regeneration by bridging large defects in a rat model.

Assessment of Bone Regeneration Using Adipose-Derived Stem Cells in Critical-Size Alveolar Ridge Defects: An Experimental Study in a Dog Model.

PURPOSE: To assess bone regeneration potential of a fibronectin- and adipose-derived stem cell-covered ceramic biomaterial in three-wall critical-size alveolar ridge defects. MATERIALS AND METHODS: In 18 dogs, four dehiscence-type and critical-size defects were created surgically in the edentulous alveolar ridge. Defects were randomly regenerated using biomaterials coated with particulate ß-tricalcium phosphate (ß-TCP), ß-TCP with fibronectin (Fn) (ß-TCP-Fn), and ß-TCP with a combination of Fn and autologous adipose-derived stem cells (ADSCs) (ß-TCP-Fn-ADSCs), leaving one defect as control. The animals were divided into three groups according to the time of euthanasia (1, 2, or 3 months of healing). RESULTS: At the time of sacrifice, statistically significant differences between the four types of defects in the total area of bone regeneration, percentage of neoformed bone matrix, medullary space, or contact between particulate biomaterial and neoformed bone matrix were not found. All defects showed a significant increase in neoformed bone matrix as sacrifice was delayed, but a uniform pattern was not followed. Only defects treated with ß-TCP-Fn-ADSCs showed a significant increase in the bone regeneration area when animals sacrificed at 3 months were compared to those sacrificed at 1 month (P = .006). CONCLUSION: The use of ADSCs in bone regeneration processes of critical-size defects of the alveolar ridge did not entail an advantage regarding greater bone regeneration as compared with other biomaterials. However, the use of ß-TCP coated with a combination of Fn and ADSCs appeared to favor stabilization of the regenerated area, allowing a more efficient maintenance of the space at 3 months of healing.

Bathysa cuspidata extract modulates the morphological reorganization of the scar tissue and accelerates skin wound healing in rats: a time-dependent study.

The technological development of pharmaceutical products based on plant extracts is currently responsible for a large number of recent innovations in healthcare. The objective of this study was to develop and investigate the effect and potential applicability of an ointment-based Bathysa cuspidata extract (BCE) for the management of skin wounds in rats. Three skin wounds of 12 mm in diameter were made on the backs of the animals, which were randomized into 4 groups according to the application received, i.e. the SAL group: 0.9% saline solution, the LAN group: lanolin, the BCE 2.5% group: 2.5% BCE emulsified in lanolin and the BCE 5% group: 5% BCE emulsified in lanolin. The applications were made daily over 21 days, and every 7 days tissue from different wounds was removed. On days 7, 14 and 21, the BCE 2.5% and BCE 5% groups showed the best results in relation to wound closure, and a higher proportion (in length, density and volume) of blood vessels and fibroblasts compared to the other groups. On days 7 and 14, there was a significant increase in the number of mast cells in these 2 groups when compared to the SAL and LAN groups. On day 21, they also had a higher proportion of collagen I than collagen III. B. cuspidata in an ointment base was effective in stimulating tissue cellularity, mast cell recruitment, neoangiogenesis, synthesis and maturation of collagen, epidermal thickness and surface area in scar tissue. These events were potentially related to the best quality and speed for skin regeneration in the rats treated with the BCE ointment.

Proliferation assessment of primary human mesenchymal stem cells on collagen membranes for guided bone regeneration.

PURPOSE: Human mesenchymal stem cells (hMSCs) hold the potential for bone regeneration because of their self-renewing and multipotent character. The goal of this study was to evaluate the influence of collagen membranes on the proliferation of hMSCs derived from bone marrow. A special focus was set on short-term eluates derived from collagen membranes, as volatile toxic materials washed out from these membranes may influence cell behavior during the short time course of oral surgery. MATERIALS AND METHODS: The proliferation of hMSCs seeded directly on a collagen membrane (BioGide) was evaluated quantitatively using the cell proliferation reagent WST-1 (4-3-[4-iodophenyl]-2-[4-nitrophenyl]-2H-[5-tetrazolio]-1, 3--benzol-disulfonate) and qualitatively by scanning electron microscopy. Two standard biocompatibility tests, namely the lactate dehydrogenase and MTT (3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazoliumbromide) tests, were performed using hMSCs cultivated in eluates from membranes incubated for 10 minutes, 1 hour, or 24 hours in serum-free cell culture medium. The data were analyzed statistically. RESULTS: Scanning electron microscopy showed large numbers of hMSCs with well-spread morphology on the collagen membranes after 7 days of culture. The WST test revealed significantly better proliferation of hMSCs on collagen membranes after 4 days of culture compared to cells cultured on a cover glass. Cytotoxicity levels were low, peaking in short-term eluates and decreasing with longer incubation times. CONCLUSION: Porcine collagen membranes showed good biocompatibility in vitro for hMSCs. If maximum cell proliferation rates are required, a prewash of membranes prior to application may be useful.

Silk fibroin conduits: a cellular and functional assessment of peripheral nerve repair.

Silk fibroin conduits were designed with appropriate porosity for peripheral nerve repair. The aim of this work was to use these conduits to examine cell inflammatory responses and functional recovery in a sciatic nerve defect model. A total of 45 randomized Lewis rats were used to create an 8-mm defect bridged by a silk guide, commercial collagen guide, or an autograft. After 1, 4, and 8 weeks, macrophage recruitment, percentage of newly formed collagen, number of myelinated axons, and gastrocnemius muscle mass were evaluated. Following 8 weeks, ED1+ cells in autograft and silk conduits decreased to <1% and 17% of week 1 values, respectively. Collagen formation revealed no difference for all measured time points, suggesting a similar foreign body response. Myelinated axon counts within the silk guide revealed a greater number of proximal spouts and distal connections than collagen guides. Gastrocnemius weights demonstrated a 27% decrease between silk and autografts after 8 weeks. This study demonstrates that, in addition to tailorable degradation rates, our silk conduits possess a favorable immunogenicity and remyelination capacity for nerve repair.

Iliac crest autogenous bone graft versus alloplastic graft and guided bone regeneration in the reconstruction of atrophic maxillae: a 5-year retrospective study on cost-effectiveness and clinical outcome.

BACKGROUND: Reconstruction of the atrophic maxillae with autogenous bone graft and jawbone-anchored bridges is a well-proven technique. However, the morbidity associated with the concept should not be neglected. Furthermore, the costs for such treatment, including general anesthesia and hospital stay, are significant. Little data are found in the literature with regard to a cost-benefit approach to various treatment alternates. PURPOSE: The aim of this retrospective study was to compare from a health-economical and clinical perspective the reconstruction of the atrophic maxillae prior to oral implant treatment either with autogenous bone grafts harvested from the iliac crest or the use of demineralized freeze-dried bone (DFDB) in combination with a thermoplastic carrier (Regeneration Technologies Inc., Alachua, FL, USA) and guided bone regeneration (GBR). MATERIALS AND METHODS: A total of 26 patients (13 + 13) were selected and matched with regard to indication, sex, and age. The study was performed 5 years after the completion of the treatment. Implant survival, morbidity, and complications were analyzed. Furthermore, a detailed analysis of the total cost for the respective treatment modality was performed, including material, costs for staff, sick leave, etc. RESULTS: The study revealed no statistical difference with regard to implant survival for the respective groups. The average total cost, per patient, for the DFDB group was 22.5% of the total cost for a patient treated with autogenous bone grafting procedures. CONCLUSIONS: The study concluded that reconstruction of atrophic maxillae with a bone substitute material (DFDB) in combination with GBR can be performed with an equal treatment outcome and with less resources and a significant reduced cost in selected cases compared with autogenous bone grafts from the iliac crest.

Assessment of cell sheets derived from human periodontal ligament cells: a pre-clinical study.

Periodontal-ligament-derived cells (PDL cells) have stem-cell-like properties and, when implanted into periodontal defects in vivo, can induce periodontal regeneration including the formation of new bone, cementum, and periodontal ligament. We have previously demonstrated that PDL cell sheets, harvested from temperature-responsive cell culture dishes, have a great potential for periodontal regeneration. The purpose of this study has been to validate the safety and efficacy of human PDL (hPDL) cell sheets for use in clinical trials. hPDL tissues from three donors were enzymatically digested, and the obtained cells were cultured with media containing autologous serum in a cell-processing center (CPC). The safety and efficacy of hPDL cell sheets were evaluated both in vitro and in vivo. In vitro studies showed that the hPDL cell sheets had high alkaline phosphatase activity and periostin expression (known PDL markers) and no contamination with microorganisms. In vivo studies revealed that hPDL cell sheets, implanted with dentin blocks, induced the formation of cementum and PDL-like tissue in immunodeficient mice. The hPDL cells presented no evidence of malignant transformation. Thus, hPDL cell sheets created in CPCs are safe products and possess the potential to regenerate periodontal tissues.

Assessment of axonal growth into collagen nerve guides containing VEGF-transfected stem cells in matrigel.

The early events associated with axonal growth into 10-mm nerve gaps were studied histologically in the rat sciatic nerve model to determine if the outgrowth of blood vessels, Schwann cells, and axons could be enhanced. In the first two experimental groups, collagen nerve guides were filled with either saline or Matrigel. Marrow-derived mesenchymal stem cells (MSCs) were added to Matrigel in two other groups, one of which contained cells transfected with VEGF (MSC/VEGF). After 21 days, the injury site was exposed, fixed, sectioned, and volume fractions of the conduit contents were determined by point counting. The bioresorbable collagen conduits appropriately guided the axons and vessels in a longitudinal direction. The volume fraction of axons was significantly greater in the group with saline when compared with all three groups with Matrigel. This measure had a significant positive correlation with actual counts of myelinated axons. The blood vessel volume fraction in the Matrigel group decreased compared with the saline group, but was restored in the MSC/VEGF group. All Matrigel groups had comparable cellularity and showed a distribution of residual Matrigel in acellular zones. The saline group, by contrast, sustained a network of delicate fibroblastic processes that compartmentalized the nerve and its natural matrix as it became infiltrated by axons as minifascicles. In conclusion, the reduction of axonal outgrowth in the Matrigel groups, when compared with the saline group, suggests that Matrigel may impede the early regenerative process even when enriched by the addition of MSCs or VEGF-transfected cells.

In vitro assessment of axonal growth using dorsal root ganglia explants in a novel three-dimensional collagen matrix.

The goal of this study was the development of a bioartificial nerve guide to induce axonal regeneration in the peripheral nervous system (PNS). In this in vitro study, the ability of a novel, 3-dimensional (3D), highly oriented, cross-linked porcine collagen scaffold to promote directed axonal growth has been studied. Collagen nerve guides with longitudinal guidance channels were manufactured using a series of chemical and mechanical treatments with a patented unidirectional freezing process, followed by freeze-drying (pore sizes 20-50 microm). Hemisected rat dorsal root ganglia (DRG) were positioned such that neural and non-neural elements could migrate into the collagen scaffold. After 21 days, S100-positive Schwann cells (SCs) migrated into the scaffold and aligned within the guidance channels in a columnar fashion, resembling "Bands of Büngner." Neurofilament-positive axons (mean length +/- SD 756 microm +/- 318 microm, maximum 1496 microm) from DRG neurons entered the scaffold where the growth within the guidance channels was closely associated with the oriented SCs. This study confirmed the importance of SCs in the regeneration process (neurotrophic theory). The alignment of SCs within the guidance channels supported directional axonal growth (contact guidance theory). The microstructural properties of the scaffold (open, porous, longitudinal pore channels) and the in vitro data after DRG loading (axonal regeneration along migrated and columnar-aligned SCs resembling "Band of Büngner") suggest that this novel oriented 3D collagen scaffold serves as a basis for future experimental regeneration studies in the PNS.

Neurobiological assessment of regenerative electrodes for bidirectional interfacing injured peripheral nerves.

Regenerative electrodes are designed to interface regenerated axons from a sectioned peripheral nerve. Applicability of regenerative electrodes depends on biocompatibility, success of axonal regeneration, secondary nerve damage, and adequacy of interface electronics. Polyimide sieve electrodes with 281 holes were chronically implanted in the severed sciatic nerve of 30 rats. Regeneration was successful in all the animals, with increasing numbers of regenerated myelinated fibers from 2 to 6 mo. However, constrictive axonopathy affected a few cases from 6 to 12 mo. postimplantation. A second electrode design with 571 holes and 27 ring electrodes was developed. The number of regenerated axons increased thanks to the larger open area. Recordings were obtained from a low proportion of electrodes on the sieve in response to distal stimulation. Difficulties for recording impulses with regenerative electrodes include the small size of regenerated axons, changes in membrane excitability and in target reconnection.

[Assessment of bovine biomaterials containing bone morphogenetic proteins bound to absorbable hydroxyapatite in rabbit segmental bone defects]. / Avaliação de biomateriais bovinos contendo proteínas morfogenéticas ósseas absorvidas a hidroxiapatita em defeitos ósseos segmentares em coelhos.

PURPOSE: To evaluate the osteo-regenerative capacity of two proprietary bone grafting materials, using a segmental defect model in both radial diaphyses of rabbits. METHODS: The right defect was filled with pooled bone morphogenetic proteins (pBMPs) bound to absorbable ultrathin powdered hydroxyapatite (HA) mixed with inorganic and demineralized bone matrix and bone-derived collagen, derived from bovine bone (Group A). The left defect was filled with bovine demineralized bone matrix and pBMPs bound to absorbable ultrathin powdered HA (Group B). In both groups, an absorbable membrane of demineralized bovine cortical was used to retain the biomaterials in the bone defects, and to guide the tissue regeneration. The rabbits were euthanized 30, 90 and 150 days after surgery. Radiographic, tomographic and histologic evaluations were carried out on all specimens. RESULTS: At 30 days, the demineralized cortical bone cover was totally resorbed in both groups. HA was totally resorbed from Group A defects, whereas HA persisted in Group B defects. A prominent foreign body reaction was evident with both products, more pronounced in sections from Group B. At 90 days, the defects in Group B exhibited more new bone than Group A. However, at 150 days after surgery, neither treatment had stimulated complete repair of the defect. CONCLUSION: The partial bone healing of the segmental defect occurred with low or none performance of the biomaterials tested.

Applications of tissue engineering in the genitourinary tract.

Congenital abnormalities and acquired disorders can lead to organ damage or loss of tissue within the genitourinary tract. For reconstructive purposes, tissue-engineering efforts are currently underway for virtually every type of tissue and organ within the urinary tract. Tissue engineering incorporates the fields of cell transplantation, materials science and engineering for the purpose of creating functional replacement tissue. This article reviews some of the principles of tissue engineering and some of the applications of these principles to the genitourinary tract.

Bioactive composite materials for tissue engineering scaffolds.

Synthetic bioactive and bioresorbable composite materials are becoming increasingly important as scaffolds for tissue engineering. Next-generation biomaterials should combine bioactive and bioresorbable properties to activate in vivo mechanisms of tissue regeneration, stimulating the body to heal itself and leading to replacement of the scaffold by the regenerating tissue. Certain bioactive ceramics such as tricalcium phosphate and hydroxyapatite as well as bioactive glasses, such as 45S5 Bioglass, react with physiologic fluids to form tenacious bonds with hard (and in some cases soft) tissue. However, these bioactive materials are relatively stiff, brittle and difficult to form into complex shapes. Conversely, synthetic bioresorbable polymers are easily fabricated into complex structures, yet they are too weak to meet the demands of surgery and the in vivo physiologic environment. Composites of tailored physical, biologic and mechanical properties as well as predictable degradation behavior can be produced combining bioresorbable polymers and bioactive inorganic phases. This review covers recent international research presenting the state-of-the-art development of these composite systems in terms of material constituents, fabrication technologies, structural and bioactive properties, as well as in vitro and in vivo characteristics for applications in tissue engineering and tissue regeneration. These materials may represent the effective optimal solution for tailored tissue engineering scaffolds, making tissue engineering a realistic clinical alternative in the near future.

An appraisal of ongoing experimental procedures in human spinal cord injury.

Clinicians and scientists in the field of spinal cord injury research and medicine are poised to begin translating promising new experimental findings into treatments for people. Advances in experimental regeneration research have led to several transplantation strategies that promote axonal regrowth and partial functional recovery in animal models of injury. In this review, we summarize current knowledge regarding various invasive experimental treatments that have been or are now being applied clinically. Various questions about the timeliness, safety, and benefits of the procedures are under discussion within the spinal cord injury (SCI) research community. We also describe guidelines for carrying out optimal clinical trials and efforts to establish specific international guidelines to translate preclinical treatment strategies into clinical trials in SCI. The clinical trial process and the role that clinical professionals have in advising individuals regarding participation in experimental procedures also is discussed.

Long term assessment of axonal regeneration through polyimide regenerative electrodes to interface the peripheral nerve.

Polyimide sieve electrodes were implanted between the severed ends of the sciatic nerve in rats. The degree of axonal regeneration through the electrode was examined by physiological and histological methods from 2 to 12 months postimplantation. Regeneration was successful in the 30 animals implanted. Functional reinnervation of hindlimb targets progressed to reach maximal levels at 6 months. Comparatively, the reinnervation of distal plantar muscles was lower than that of proximal muscles and of digital nerves. The number of regenerated myelinated fibers increased from 2 to 6 months, when it was similar to control values. The majority of myelinated fibers crossing the via holes and regenerated through the distal nerve had a normal appearance. However, in a few cases decline of target reinnervation and loss of regenerated nerve fibers was found from 6 to 12 months postimplantation. Motor axons labeled by ChAT immunoreactivity regenerated scattered within minifascicles, although they were found at higher density at the periphery of the regenerated nerve. The number of ChAT-positive axons was markedly lower distally than proximally to the sieve electrode.

Plant regeneration of indica rice (Oryza sativa) cultivars from mature embryo-derived calli.

Plant regeneration from seven-week-old callus cultures derived from mature embryos of several indica rice cultivars was achieved with frequencies of morphogenic calli from 10 to 47%. Three media were tested both for callogenesis and plant regeneration. For 3 of the 7 genotypes examined, the best combination of media for plant regeneration was Murashige & Skoog basal medium: MSC (callogenesis) and MSR (regeneration). The rates of callogenesis were not related to the capacity for plant regeneration. Two genotypes CR-1113 and CR-5272 produced the highest number of regenerated green plants. The results of this study suggest that genetic differences could be directly linked to the ability to regenerate in these plant cultivars.

Plant regeneration of indica rice (Oryza sativa) cultivars from mature embryo-derived calli

Plant regeneration from seven-week-old callus cultures derived from mature embryos of several indica rice cultivars was achieved with frequencies of morphogenic calli from 10 to 47 percent. Three media were tested both for callogenesis and plant regeneration. For 3 of the 7 genotypes examined, the best combination of media for plant regeneration was Murashige & Skoog basal medium: MSC (callogenesis) and MSR (regeneration). The rates of callogenesis were not related to the capacity for plant regeneration. Two genotypes CR-1113 and CR-5272 produced the highest number of regenerated green plants. The results of this study suggest that genetic differences could be directly linked to the ability to regenerate in these plant cultivars