Effect of Intratracheal Administration of Adipose-derived Stromal Cells on Bleomycin-induced Lung Injury in a Rat Model.

BACKGROUND: Mesenchymal stromal cells (MSCs) have been intensively investigated in regenerative medicine. Among the different types of MSCs, adipose tissue-derived stromal cells (ASCs) can be obtained with relatively less invasive techniques. Since ASC administration is a candidate strategy for the treatment of refractory diseases including pulmonary fibrosis, we investigated whether intratracheal injection of ASCs had therapeutic potential against bleomycin (BLM)-induced lung injury in rats. METHODS: BLM was intratracheally administered to rats, and 1 week later ASCs were harvested. Two weeks after BLM treatment, ASCs or phosphate-buffered saline (PBS) were injected autologously into the rats via the trachea A semi-quantitative histological evaluation was conducted to assess the injured lungs, followed by cell tracing at 3 or 6 weeks after BLM instillation. RESULTS: ASC administration did not affect the severity of lung damage on the third week after BLM exposure, but prevented further aggravation of the lung injury, as apparent on the sixth week. A fluorescent cell tracer revealed that the majority of ASCs did not appear to have penetrated inside the lung region injured by BLM on the third week after BLM instillation, but some of these cells sprouted deep into the thick distorted architecture of the injured lung on the sixth week after the BLM instillation. CONCLUSIONS: The results of the present study suggest that ASCs may play a role in the prevention of ongoing aggravation of lung injury in the long term.

Adipose-derived stem cells enhance tissue regeneration of gastrotomy closure.

BACKGROUND: The aim of this study was to examine whether transplantation of adipose-derived stem cells (ADSCs) improves healing of a gastrotomy closure in rats. In digestive surgery, anastomotic leakage is a serious postoperative complication and anastomotic stenosis may reduce quality of life. Recent studies have suggested that ADSCs play material roles in intestinal healing, acceleration of angiogenesis, and reduction of fibrosis, and treatment with ADSCs may improve healing. MATERIALS AND METHODS: ADSCs were isolated from intra-abdominal white adipose tissue of 40 male Wistar rats (weight 300 g) in four groups (n = 10 each). Gastrotomy closures were prepared surgically in all rats. Controls were treated with phosphate-buffered saline injection and sacrificed 7 d (group 1) or 28 d (group 3) after the surgery. Other animals were treated with locally autotransplanted ADSCs (labeled by CM-DiI) and sacrificed 7 d (group 2) or 28 d (group 4) after the surgery. Histopathologic features were evaluated in the four groups. RESULTS: Injection of ADSCs significantly enhanced angiogenesis and collagen deposition after 7 d, indicating improved healing of the gastrotomy closure. In contrast, ADSC transplantation significantly reduced collagen deposition after 28 d. The bursting pressure was higher in the transplant groups after 7 d. CONCLUSIONS: ADSCs enhance tissue regeneration in gastrotomy closures by accelerating angiogenesis and fibrosis in the early healing period. In the late period, ADSCs prevent excessive fibrosis and assist in regeneration of tissues that closely resemble the native structure. These results suggest that therapy with transplanted ADSCs might improve postoperative complications in digestive surgery.

Intrahepatic biliary ablation with pure ethanol: an experimental model of biliary atresia.

PURPOSE: We describe a new rat model of biliary atresia, induced by biliary ablation with pure ethanol. METHODS: A catheter was inserted and fixed in the common bile duct of male rats. Saline or pure ethanol was injected through the catheter and the animals were monitored for 8 weeks thereafter. We measured total bilirubin (T-Bil), aspartate aminotransferase (AST), alanine transaminase (ALT), and hyaluronic acid (HA) and examined liver biopsy specimens immunohistochemically for α-smooth muscle actin staining (α-SMA) and transforming growth factor-ß1 (TGF-ß1). RESULTS: The ethanol injection group animals were further divided into a temporary and a persistent liver dysfunction group. In the persistent group, T-Bil, AST, ALT and HA levels were significantly higher after 8 weeks in the persistent group than in the control group and the temporary group. In the ethanol injection group, α-SMA expression was prominent in the surrounding proliferative bile ducts and portal areas. The distribution of TGF-ß1 was found prominently in hepatocytes in the center of nodules and in ductular epithelial cells. CONCLUSIONS: This study characterizes the effects of ethanol-induced bile duct injury in rats, resulting in sclerosing cholangitis and its secondary effects. We believe that this experimental model will prove useful in the study of biliary atresia.

Accelerated generation of human induced pluripotent stem cells with retroviral transduction and chemical inhibitors under physiological hypoxia.

Induced pluripotent stem (iPS) cells are generated from somatic cells by the forced expression of a defined set of pluripotency-associated transcription factors. Human iPS cells can be propagated indefinitely, while maintaining the capacity to differentiate into all cell types in the body except for extra-embryonic tissues. This technology not only represents a new way to use individual-specific stem cells for regenerative medicine but also constitutes a novel method to obtain large amounts of disease-specific cells for biomedical research. Despite their great potential, the long reprogramming process (up to 1month) remains one of the most significant challenges facing standard virus-mediated methodology. In this study, we report the accelerated generation of human iPS cells from adipose-derived stem (ADS) cells, using a new combination of chemical inhibitors under a setting of physiological hypoxia in conjunction with retroviral transduction of Oct4, Sox2, Klf4, and L-Myc. Under optimized conditions, we observed human embryonic stem (ES)-like cells as early as 6 days after the initial retroviral transduction. This was followed by the emergence of fully reprogrammed cells bearing Tra-1-81-positive and DsRed transgene-silencing properties on day 10. The resulting cell lines resembled human ES cells in many respects including proliferation rate, morphology, pluripotency-associated markers, global gene expression patterns, genome-wide DNA methylation states, and the ability to differentiate into all three of the germ layers, both in vitro and in vivo. Our method, when combined with chemical inhibitors under conditions of physiological hypoxia, offers a powerful tool for rapidly generating bona fide human iPS cells and facilitates the application of iPS cell technology to biomedical research.

Use of adipose tissue-derived stromal cells for prevention of esophageal stricture after circumferential EMR in a canine model.

BACKGROUND: EMR is an accepted treatment for early esophageal carcinoma. However, resection of a large mucosal area often causes postoperative esophageal stricture. OBJECTIVE: To investigate the efficacy of autologous adipose tissue-derived stromal cells (ADSCs) for prevention of stricture formation after EMR in dogs. DESIGN: Animal study. SETTING: University research center. INTERVENTION: Ten beagle dogs were randomized into a control group and an ADSCs-injected (ADSC) group. The ADSCs were isolated from autologous adipose tissue. Immediately after circumferential esophageal EMR, about 5 × 10(6) ADSCs suspended in 8 mL of phosphate-buffered saline solution were injected endoscopically into the residual submucosa of the ADSC group, whereas the control group received only 8 mL of phosphate-buffered saline solution. MAIN OUTCOME MEASUREMENTS: Dysphagia score, weight loss, rate of mucosal constriction, and histologic assessments. RESULTS: In the control and ADSC groups, the median dysphagia scores were 4 and 1 (P < .043), the mean degrees of mucosal constriction were 75.7% and 45.3% (P < .008), and the numbers of nascent microvessels in the submucosal layer were 7.4 and 16.2 per unit area (P = .007), respectively. Atrophy and fibrosis of the muscularis propria layer were observed in the control group. LIMITATIONS: Animal study, small sample size. CONCLUSION: Injection therapy with autologous ADSCs suppresses constriction of the esophageal mucosa and improves clinical symptoms after circumferential EMR in this canine model.

Bone regeneration utilizing a newly developed octacalcium phosphate/weakly denatured collagen scaffold.

INTRODUCTION: Autologous bone grafting or various bone-regenerating materials are used to treat bone defects caused by tumor resection and accident trauma. Octacalcium phosphate, a reasonable bone regenerative material, activates osteoblasts. We fabricated a composite material, octacalcium phosphate/weakly denatured collagen, as a new scaffold. We aimed to investigate the osteoregenerative effect of the octacalcium phosphate/weakly denatured collagen scaffold (compared with that of weakly denatured collagen) in skull defects in a canine model. METHODS: Atelocollagen was extracted from porcine skin via pepsin treatment. The weakly denatured collagen scaffold was fabricated with a freeze-dried and thermally crosslinked atelocollagen suspension at pH 7.4. Octacalcium phosphate was synthesized using Ca-acetate and NaH2PO4. Octacalcium phosphate particles (diameter, 199-298 µm) were mixed with a collagen matrix to fabricate an octacalcium phosphate/weakly denatured collagen scaffold. Bilateral defects (diameter, 10 mm; full-thickness) were induced in dog skulls, and the octacalcium phosphate/weakly denatured collagen and weakly denatured collagen scaffolds were implanted into the defects. RESULTS: Eight weeks after implantations, bone regeneration was evaluated via histopathological analysis. It revealed osteoblast infiltration and osteoregeneration in all defects treated with the octacalcium phosphate/weakly denatured collagen scaffold. The defects treated with weakly denatured collagen scaffold or without any scaffold mostly contained connective tissue, with no neo-osteogenesis. DISCUSSION: The novel octacalcium phosphate/weakly denatured collagen scaffold better promotes osteoregeneration than the weakly denatured collagen scaffold; this "in situ tissue engineering" approach is potentially clinically applicable for bone reconstruction.

Regeneration of central nervous tissue using a collagen scaffold and adipose-derived stromal cells.

Adipose-derived stromal cells (ASCs) include stem cells, which have the potential to differentiate into a variety of cell lineages. The regeneration of central nerves was examined using ASCs and a collagen scaffold. A cerebral cortex defect (3 x 4 x 3 mm(3)) was created in the left frontal lobe of 16 male rats. In one group (n = 8), collagen (3 x 4 x 3 mm(3)) seeded with DiI-labeled ASCs was implanted in the defect. In order to seed the ASCs, a combination of the rotary cell culture system and pressing the collagen scaffold gently several times with a glass rod was applied. In the control group (n = 8), collagen was implanted without ASCs. The rats were sacrificed at 1 month after the scaffold implantation. Histologically, 0.2% of the implanted ASCs were positive for anti-human/rat microtubule-associated protein 2 (MAP2) antibody and microvessels were present at a density of 4.6 +/- 1.2/mm(2) within the collagen scaffold-implanted area in each coronal section. In the control group, no MAP2-positive cells were detected and the microvessel density was 0.6 +/- 0.4/mm(2). These data suggest that ASCs seeded into a collagen scaffold may have the potential to promote regeneration of nervous tissue after cerebral cortex injury.

Can nerve regeneration on an artificial nerve conduit be enhanced by ethanol-induced cervical sympathetic ganglion block?

This study aimed to determine whether nerve regeneration by means of an artificial nerve conduit is promoted by ethanol-induced cervical sympathetic ganglion block (CSGB) in a canine model. This study involved two experiments-in part I, the authors examined the effect of CSGB by ethanol injection on long-term blood flow to the orofacial region; part II involved evaluation of the effect of CSGB by ethanol injection on inferior alveolar nerve (IAN) repair using polyglycolic acid-collagen tubes. In part I, seven Beagles were administered left CSGB by injection of 99.5% ethanol under direct visualization by means of thoracotomy, and changes in oral mucosal blood flow in the mental region and nasal skin temperature were evaluated. The increase in blood flow on the left side lasted for 7 weeks, while the increase in average skin temperature lasted 10 weeks on the left side and 3 weeks on the right. In part II, fourteen Beagles were each implanted with a polyglycolic acid-collagen tube across a 10-mm gap in the left IAN. A week after surgery, seven of these dogs were administered CSGB by injection of ethanol. Electrophysiological findings at 3 months after surgery revealed significantly higher sensory nerve conduction velocity and recovery index (ratio of left and right IAN peak amplitudes) after nerve regeneration in the reconstruction+CSGB group than in the reconstruction-only group. Myelinated axons in the reconstruction+CSGB group were greater in diameter than those in the reconstruction-only group. Administration of CSGB with ethanol resulted in improved nerve regeneration in some IAN defects. However, CSGB has several physiological effects, one of which could possibly be the long-term increase in adjacent blood flow.

Regeneration of gingival tissue using in situ tissue engineering with collagen scaffold.

OBJECTIVE: The aim of the study was to evaluate 2 types of collagen scaffold for gingival regeneration. STUDY DESIGN: Two types of collagen scaffolds, CS-pH7.4 and CS-pH3.0, were prepared by processing atelocollagen at pH 7.4 or 3.0, respectively, followed by dehydrothermal treatment. Gingival wounds with sizes of 4 × 6 mm (rectangle) or 6 mm diameter (circle) were made with buccal incisions in beagle dogs. The defective area was surgically covered with the CS-pH7.4, CS-pH3.0, or no scaffold (control). Gingival regeneration was assessed by monitoring the differences in the lengths of the epithelial and submucosal tissues at the wound site and the normal site. Histopathologic assessments were performed by 4 evaluators independently; statistical significance was evaluated by using the Wald test. RESULTS: Significantly higher recovery of epithelial and submucosal tissues, which, in turn, resulted in recovery of gum thickness, was observed in gingival wounds treated with the CS-pH7.4 compared with that in the control. CS-pH3.0 treatment also resulted in higher gingival regeneration compared with the control; however, the effects were more pronounced in wounds treated with the CS-pH7.4. CS-pH7.4-treated wounds showed better gingival regeneration compared with the control and CS-pH3.0-treated wounds, even after adjusting for interevaluator differences using a linear mixed model. CONCLUSIONS: CS-pH7.4 is a promising scaffold for gingival tissue regeneration.

Optimal dehydrothermal processing conditions to improve biocompatibility and durability of a weakly denatured collagen scaffold.

Collagen scaffolds are essential for tissue regeneration; however, preprocessing of these scaffolds is necessary because of their poor mechanical properties. The aim of this study was to determine the optimal condition for preparing a collagen scaffold with biocompatibility and durability. An atelocollagen fiber suspension was made and stored at -10°C in a container that could be cooled from the bottom to provide an orientation perpendicular to the collagen fiber and facilitate cell infiltration into the scaffold. After freeze-drying the frozen suspension, various collagen scaffolds were made by dehydrothermal (DHT) treatment under different conditions (processing temperature: 120-160°C for 0-28 h). Sections of the obtained materials were embedded under the back skin of rats, and the thickness and biocompatibility of the residual scaffold were evaluated after 2 weeks. The number of foreign body giant cells was counted to evaluate biocompatibility. Although the residual scaffold was thick, excessive DHT treatment caused a strong foreign body reaction. Weak DHT treatment resulted in a collagen scaffold with good biocompatibility but with reduced thickness. Overall, these results showed the restricted optimal conditions to make a collagen scaffold with good biocompatibility and ability to maintain sufficient space for tissue regeneration. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 2301-2307, 2017.

Experimental study on the regeneration of peripheral nerve gaps through a polyglycolic acid-collagen (PGA-collagen) tube.

We have developed a bioabsorbable polyglycolic acid (PGA) tube filled with collagen sponge (PGA-collagen tube) as a nerve connective guide, and compared its effectiveness with that of autograft in terms of nerve regeneration across a gap. The PGA-collagen tube was implanted into 24 beagle dogs across a 15-mm gap in the left peroneal nerve. The right peroneal nerve was reconstructed with the autograft harvested from the left side, as a control. After the surgery, the connective tissue extended from both cut ends in the PGA-collagen tube and connected again at the center. Pathologically, the collagen sponge in the tube provided adequate scaffolding for nerve tissue extension, and the nerve tissue reconnected within 3 weeks. Electrophysiologically, muscle-evoked potentials (MEPs) and compound nerve action potentials (CNAPs) were detected 18 days after the surgery. For up to 6 months postsurgery, CNAPs and somatosensory-evoked potentials (SEPs) on the PGA-collagen side had a shorter latency and larger peak voltage than those on the autograft side. The myelinated axons on the PGA side were larger in diameter than those on the autograft side. It is suggested that the PGA-collagen tube has the potential to be an effective alternative to conventional autografting for the repair of some peripheral nerve defects.

Observation of the esophagus, pharynx and lingual root by gastrointestinal endoscopy with a percutaneous retrograde approach.

AIM: To evaluate the efficacy of retrograde observation of the esophagus, pharynx, larynx and lingual root. METHODS: With the beagle dog under anesthesia, the anterior wall of the stomach was fixed on the abdominal wall in a similar way to percutaneous endoscopic gastrostomy. The gastrointestinal scope was inserted via a 12 mm laparoscopic port for subsequent retrograde observation from stomach to the oral cavity. RESULTS: With this technique, direct observation of gastric cardia was possible without restriction. The cervical esophagus was dilated well, also allowing clear observation of the hypopharyngo-esophageal junction. If the tongue was manually pulled out forward, observation of the lingual root was possible. CONCLUSION: This procedure is easy and effective for pre-treatment evaluation of the feasibility of endoscopic resection in cases of superficial carcinoma of head and neck.

Development of new nerve guide tube for repair of long nerve defects.

A novel nerve guide tube (poly (L-lactic) acid (PLLA)/ polyglycolic acid (PGA)-c-tube) capable of repairing long peripheral nerve injuries in a canine model has been developed. The tube was created by braiding together PLLA and PGA and then coating it with collagen. PLLA was newly added to the formulation to achieve higher sustainability. The tube was compared with a PGA-collagen tube in clinical use since 2002 having the same structure with a collagen coating but composed of PGA alone (PGA-c-tube). When tested for repair of a 40-mm gap in the left peroneal nerve, using PLLA/PGA-c-tube (n = 15), PGA-c-tube (n = 15), and a negative control group where the cut stump was capped using a silicone cap (n = 15), the lumen structure essential for securing the space for nerve regeneration was maintained in PLLA/PGA-c-tube for over 12 months with a higher number of axons both within the tube and at the distal nerve end. Electrophysiological evaluation revealed that the amplitude of compound muscle action potentials and sensory nerve action potentials after nerve regeneration with PLLA/PGA-c-tube were significantly higher. When assessed using magnetic resonance imaging (MRI), the volume of the tibialis anterior (TA) muscle in dogs that had undergone nerve repair using PLLA/PGA-c-tube was approximately 80% that of the positive control at 12 months. Functional analysis conducted by assessing the ankle angle revealed faster recovery in the PLLA/PGA-c-tube group. Better regeneration was achieved using a PLLA/PGA-c-tube that contains the slowly decomposing fiber material, PLLA. This indicates potential for repair of even longer nerve gaps or defects located near joints, and also clinical application.

In situ tissue engineering for tracheal reconstruction using a luminar remodeling type of artificial trachea.

BACKGROUND: After successful trials of tracheal reconstruction using mesh-type prostheses in canine models, the technique has been applied clinically to human patients since 2002. To enhance tissue regeneration, we have applied a new tissue engineering approach to this mesh-type prosthesis. METHODS: The prosthesis consists of a polypropylene mesh tube reinforced with a polypropylene spiral and atelocollagen layer. The cervical tracheas of 18 beagle dogs were replaced with the prosthesis. The collagen layer was soaked with peripheral blood in 6 of the dogs, with bone marrow aspirate in another 6, and with autologous multipotential bone marrow-derived cells (mesenchymal stem cells) in another 6. The dogs were humanely killed at 1 to 12 months after the operation. RESULTS: All 18 dogs survived the postoperative period. Bronchoscopically, 3 of 4 dogs in the peripheral blood group showed stenosis, whereas no stenosis was evident in all 8 of the dogs in the bone marrow and mesenchymal stem cell groups 6 months after the operation. Faster epithelialization and fewer complications, such as mesh exposure and luminal stenosis, were observed in these two groups than in the peripheral blood group. Histologically, the cells from autologous bone marrow were found to proliferate into the tracheal tissue during the first month. Cilial movement in these two groups was faster than that in the peripheral blood group and recovered to 80% to 90% of the normal level. CONCLUSIONS: Bone marrow aspirate and mesenchymal stem cells enhance the regeneration of the tracheal mucosa on this prosthesis. This in situ tissue engineering approach may facilitate tracheal reconstruction in the clinical setting.

Experimental repair of phrenic nerve using a polyglycolic acid and collagen tube.

OBJECTIVE: The feasibility of a nerve guide tube for regeneration of the phrenic nerve with the aim of restoring diaphragmatic function was evaluated in a canine model. METHODS: The nerve tube, made of woven polyglycolic acid mesh, had a diameter of 3 mm and was filled with collagen sponge. This polyglycolic acid-collagen tube was implanted into a 10-mm gap created by transection of the right phrenic nerve in 9 beagle dogs. The tubes were implanted without a tissue covering in 5 of the 9 dogs (group I), and the tubes were covered with a pedicled pericardial fat pad in 4 dogs (group II). Chest x-ray films, muscle action potentials, and histologic samples were examined 4 to 12 months after implantation. RESULTS: All of the dogs survived without any complications. x-ray film examination showed that the right diaphragm was paralyzed and elevated in all dogs until 3 months after implantation. At 4 months, movement of the diaphragm in the implanted side was observed during spontaneous breathing in 1 dog of group I and in 3 dogs of group II. In the dogs showing diaphragm movement, muscle action potentials were evoked in the diaphragm muscle, indicating restoration of nerve function. Regeneration of the phrenic nerve structure was also examined on the reconstructed site using electron microscopy. CONCLUSION: The polyglycolic acid-collagen tube induced functional recovery of the injured phrenic nerve and was aided by coverage with a pedicled pericardial fat pad.

Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis.

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.