Adipose tissue is derived from the mesoderm during embryonic development and is present in every mammalian species, located throughout the body. Adipose tissue serves as an endocrine organ, functioning to maintain energy metabolism through the storage of lipids. While two types of adipose tissue exist (brown and white), white adipose yields the commonly studied adipose-derived stem cells (ASCs). Adipose-derived stem cells provide a promising future in the field of tissue engineering and regenerative medicine. Due to their wide availability and ability to differentiate into other tissue types of the mesoderm-including bone, cartilage, muscle, and adipose-ASCs may serve a wide variety of applications. Adipose stem cells have been utilized in studies addressing osteoarthritis, diabetes mellitus, heart disease, and soft tissue regeneration and reconstruction after mastectomy and facial repair. Various delivery systems and scaffolds to incorporate adipose stem cells have also been established. Adipose stem cells have been studied in vitro and in vivo. Much information in vitro has been obtained on adipose stem cell potency and biology as a function of donor gender, body mass index, and anatomical location. Further in vitro studies have examined the various cell populations within the heterogeneous population within the stromal vascular fraction (SVF) from which ASCs are obtained. While many animal models are used to investigate adipose tissue, preclinical in vivo experiments are most widely conducted in the mouse model. Common analyses of animal studies utilizing ASCs include pre-labeling cells and immunostaining cells.
Severe injuries to peripheral nerves are challenging to repair. Standard-of-care treatment for nerve gaps >2 to 3 centimeters is autografting; however, autografting can result in neuroma formation, loss of sensory function at the donor site, and increased operative time. To address the need for a synthetic nerve conduit to treat large nerve gaps, we investigated a biodegradable poly(caprolactone) (PCL) conduit with embedded double-walled polymeric microspheres encapsulating glial cell line–derived neurotrophic factor (GDNF) capable of providing a sustained release of GDNF for >50 days in a 5-centimeter nerve defect in a rhesus macaque model. The GDNF-eluting conduit (PCL/GDNF) was compared to a median nerve autograft and a PCL conduit containing empty microspheres (PCL/Empty). Functional testing demonstrated similar functional recovery between the PCL/GDNF-treated group (75.64 ± 10.28%) and the autograft-treated group (77.49 ± 19.28%); both groups were statistically improved compared to PCL/Empty-treated group (44.95 ± 26.94%). Nerve conduction velocity 1 year after surgery was increased in the PCL/GDNF-treated macaques (31.41 ± 15.34 meters/second) compared to autograft (25.45 ± 3.96 meters/second) and PCL/Empty (12.60 ± 3.89 meters/second) treatment. Histological analyses included assessment of Schwann cell presence, myelination of axons, nerve fiber density, and g-ratio. PCL/GDNF group exhibited a statistically greater average area occupied by individual Schwann cells at the distal nerve (11.60 ± 33.01 μm2) compared to autograft (4.62 ± 3.99 μm2) and PCL/Empty (4.52 ± 5.16 μm2) treatment groups. This study demonstrates the efficacious bridging of a long peripheral nerve gap in a nonhuman primate model using an acellular, biodegradable nerve conduit.
Biodegradable magnesium metal filaments placed inside biodegradable nerve conduits might provide the physical guidance support needed to improve the rate and extent of regeneration of peripheral nerves across injury gaps. In this study, we examined basic issues of magnesium metal resorption and biocompatibility by repairing sub-critical size gap injuries (6 mm) in one sciatic nerve of 24 adult male Lewis rats. Separated nerve stumps were connected with poly(caprolactone) nerve conduits, with and without magnesium filaments (0.25 mm diameter, 10 mm length), with two different conduit filler substances (saline and keratin hydrogel). At 6 weeks after implantation, magnesium degradation was examined by micro-computed tomography and histological analyses. Magnesium degradation was significantly greater when the conduits were filled with an acidic keratin hydrogel than with saline (p < 0.05). But magnesium filaments in some animals remained intact for 6 weeks. Using histological and immunocytochemical analyses, good biocompatibility of the magnesium implants was observed at 6 weeks, as shown by good development of regenerating nerve mini-fascicles and only mild inflammation in tissues even after complete degradation of the magnesium. Nerve regeneration was not interrupted by complete magnesium degradation. An initial functional evaluation, determination of size recovery of the gastrocnemius muscle, showed a slight improvement due to magnesium with the saline but not the keratin filler, compared with respective control conduits without magnesium. These results suggest that magnesium filament implants have the potential to improve repair of injured peripheral nerve defects in this rodent model.
A current clinical challenge is to replace autografts for repair of injury gaps in peripheral nerves, which can occur due to trauma or surgical interruption. Biodegradable metallic magnesium filaments, placed inside hollow nerve conduits, could support nerve repair by providing contact guidance support for axonal regeneration. This was tested by repairing sciatic nerves of adult rats with single magnesium filaments placed inside poly(caprolactone) nerve conduits. Controls were empty conduits, conduits containing titanium filaments and/or isografts from donor rats. With a nerve gap of 6 mm and 6 weeks post-repair, magnesium filaments had partially resorbed. Regenerating cells had attached to the filaments and axons were observed in distal stumps in all animals. Axon parameters were improved with magnesium compared to conduits alone or conduits with single titanium filaments. With a longer gap of 15 mm and 16 weeks post-repair, functional parameters were improved with isografts, but not with magnesium filaments or empty conduits. Magnesium filaments were completely resorbed and no evidence of scarring was seen. While axon outgrowth was not improved with the longer gap, histological measures of the tissues were improved with magnesium compared to empty conduits. Therefore, the use of magnesium filaments is promising because they are biocompatible and improve aspects of nerve regeneration. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3148-3158, 2017.
Background Biomedical implants used in tissue engineering repairs, such as scaffolds to repair peripheral nerves, can be too large to examine completely with histological analyses. Micro-computed tomography (micro-CT) with contrast agents allows ex vivo visualization of entire biomaterial implants and their interactions with tissues, but contrast agents can interfere with histological analyses of the tissues or cause shrinkage or loss of antigenicity. New Method Soft tissue, ex vivo micro-CT imaging using Lugol’s iodine was compatible with histology after using a rapid (48 h) method of removing iodine. Results Adult normal and repaired rat sciatic nerves were infiltrated ex vivo with iodine, imaged with micro-CT and then the iodine was removed by incubating tissues in sodium thiosulfate. Subsequent paraffin sections of normal nerve tissues showed no differences in staining with hematoxylin and eosin or immunostaining with multiple antibodies. Iodine treatment and removal did not alter axonal diameter, nuclear size or relative area covered by immunostained axons (p>0.05). Combining imaging modalities allowed comparisons of macroscopic and microscopic features of nerve tissues regenerating through simple nerve conduits or nerve conduits containing a titanium wire for guidance. Comparison with Existing Methods; Quantification showed that treatment with iodine and sodium thiosulfate did not result in tissue shrinkage or loss of antigenicity. Conclusions Because this combination of treatments is rapid and does not alter tissue morphology, this expands the ex vivo methods available to examine the success of biomaterial implants used for tissue engineering repairs.
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