Collagenase Digestion Research Articles

Synthesis, characterization and application of biodegradable polymer grafted novel bioprosthetic tissue

Animal tissue has an extended history of clinical use in applications like heart valve bioprosthesis devices, cardiovascular surgical applications etc. but often does not last long after implantation in the body due to rapid unwanted degradation. The goal of this work is to develop novel composite biomaterials by grafting biological tissue with synthetic, biodegradable polymers. In the current research phase, porcine submucosa, ureter and bovine pericardial tissue are grafted with poly DL-lactide (PLA), poly glycolide (PGA) and poly DL-lactide glycolide (PLGA) copolymers. The grafted and control tissues are characterized by FTIR and SEM. The biodegradability of the tissue-graft composite materials is determined by pepsin and collagenase digestion assays, showing it can be tailored by varying the grafted polymer type and amount. The grafted tissues can be tuned for a particular clinical or tissue engineering applications including drug delivery with little or no burst release and sustained/controlled delivery.

Mice dental pulp and periodontal ligament endothelial cells exhibit different proangiogenic properties.

Dental pulp is a highly vascularized tissue with a high regenerative capacity. This is attributed to its unique blood supply and the presence of progenitor or postnatal dental pulp stem cells. Here we aimed to isolate and compare the angiogenic properties of endothelial cells (EC) prepared from mouse dental pulp and periodontal ligament (PDL). EC were isolated from 4-week-old wild type immorto mice. Mice were sacrificed and after mandible isolation, the molar and incisor teeth and the PDL from molar teeth were dissected. EC were prepared by collagenase digestion of tissues and affinity purification using magnetic beads coated with platelet/endothelial cell adhesion molecule-1 (PECAM-1/CD31) antibody. EC prepared from incisor and molar pulps and PDL were examined for expression of appropriate markers by fluorescence-activated cell sorting (FACS) analysis. The proliferation, migration, and capillary morphogenesis of EC were evaluated. Ex vivo sprouting angiogenesis from various tissues was also compared. Data were analyzed at the level of significance of P<0.05. Pulp EC prepared from incisors proliferated and migrated significantly faster than molar and PDL EC (P<0.05). In addition, molar and PDL EC formed a more extensive capillary network when plated on Matrigel. This is consistent with the lower proliferative and migratory characteristics of these cells compared with incisor EC (P<0.05). However, PDL tissue showed significantly more sprouting area than molar and incisor pulp tissues (P<0.05). Thus, pulp EC from molar and incisor and PDL EC present different proangiogenic properties. Collectively our results suggest that EC from different tooth tissue have unique characteristics related to their target tissue and function.

Subacute effects of rose Bengal/Green light cross linking on rabbit thin corneal stability and safety.

The purpose of this study was to investigate the subacute effects of Rose Bengal (RB) and 532 nm green light-induced photochemical crosslinking (RB-PCL) on rabbit thin corneal stability and safety in vivo. Rabbit thin corneal models with 250 μm thickness were created by photorefractive keratectomy surgery. Photochemical crosslinking with green light (wavelength 532 nm) at an illumination intensity of 0.4 W/cm2 for 250 s (100 J/cm2 ) was performed, followed by antibiotic treatment and slit lamp monitoring for four weeks. At the end of week four, corneal biomechanical stiffness, biochemical resistance to collagenase digestion, and corneal cellular morphology were assessed. The penetration depth of RB into the corneal stromal was measured by confocal microscopy. At the end of week 4, RB-PCL had increased corneal tensile strength by an average 2.5-fold and had extended the corneal collagenase digestion time from 10.17 ± 2.93 to 15.83 ± 2.64 days. RB penetrated approximately 90 µm into the corneal stroma. RB-PCL did not alter the corneal endothelial and stromal morphology at the cellular or subcellular levels, according to electron microscopic examination. RB and 532 nm green light irradiation effectively induced crosslinking in rabbit thin cornea, by increasing both the biomechanical stiffness and the biochemical resistance without evidence of morphological damage to the corneal endothelium or stroma. This study demonstrated the efficacy of RB-PCL in strengthening thin cornea at four weeks after the treatment, providing a potential and possibly better option for treating corneal ectasia disorders in cases where corneal thickness is less than 400 µm. Lasers Surg. Med. 50:324-332, 2018. © 2017 Wiley Periodicals, Inc.

Establishment and long-term culture of human cystic fibrosis endothelial cells

Endothelial cell (EC) dysfunction has been reported in cystic fibrosis (CF) patients. Thus, the availability of CF EC is paramount to uncover mechanisms of endothelial dysfunction in CF. Using collagenase digestion, we isolated cells from small fragments of pulmonary artery dissected from non-CF lobes or explanted CF lungs. These cells were a heterogeneous mixture, containing variable percentages of EC. To obtain virtually pure pulmonary artery endothelial cells (PAEC), we developed an easy, inexpensive, and reliable method, based on the differential adhesion time of pulmonary artery cells collected after collagenase digestion. With this method, we obtained up to 95% pure non-CF and CF-PAEC. Moreover, we also succeed at immortalizing both PAEC and CF-PAEC, which remained viable and with unchanged phenotype and proliferation rate over the 30th passage. These cells recapitulated cystic fibrosis transmembrane conductance regulator expression and functions of the parental cells. Thus, we isolated for the first time endothelial cells from CF patients, providing a valuable tool to define the emerging role of EC in CF lung and vascular disease.

Acetic acid and pepsin result in high yield, high purity and low macrophage response collagen for biomedical applications

Collagen based devices are frequently associated with foreign body response. Although several pre- (e.g. species, state of animal, tissue) and post- (e.g. cross-linking, scaffold architecture) extraction method factors have a profound effect on foreign body response, little is known about which and how during the extraction process factors mediate foreign body response. In this study, we assessed the influence of acetic acid and hydrochloric acid and the utilisation or not of pepsin or salt precipitation during collagen extraction on the yield, purity, free amines, denaturation temperature, resistance to collagenase degradation and macrophage response. Acetic acid/pepsin extracted collagen exhibited the highest yield, purity and free amine content and the lowest denaturation temperature. No differences in resistance to collagenase digestion were detected between the groups. Although all treatments exhibited similar macrophage morphology comprised of round cells (M1 phenotype), elongated cells (M2 phenotype) and cell aggregates (foreign body response), significantly more elongated cells were observed on HC films. Although no differences in metabolic activity were observed between the groups, the DNA concentration was significantly lower for the hydrochloric acid treatments. Further, cytokine analysis revealed that hydrochloric acid treatments induced significantly higher IL-1β and TNF-α release with respect to acetic acid treatments. Salt precipitation did not influence the parameters assessed. Collectively, these data suggest that during the collagen extraction process variables should also be monitored as, evidently, they affect the physicochemical and biological properties of collagen preparations.

Effects of microRNA-140 gene transfection with nucleus localization signal linked nucleic kinase substrate short peptide conjugated chitosan on rabbit articular chondrocytes

To investigate the effects of nucleus localization signal linked nucleic kinase substrate short peptide (NNS) conjugated chitosan (CS) ( NNSCS) mediated the transfection of microRNA-140 (miR-140) in rabbit articular chondrocytes in vitro. Recombinant plasmid GV268-miR-140 and empty plasmid GV268 were combined with NNSCS to form NNSCS/pDNA complexes, respectively. Chondrocytes were isolated and cultured through trypsin and collagenase digestion from articular cartilage of newborn New Zealand white rabbits. The second generation chondrocytes were divided into 3 intervention groups: normal cell control group (group A), NNSCS/GV268 empty plasmid transfection group (group B), and NNSCS/GV268-miR-140 transfection group (group C). NNSCS/GV268 and NNSCS/GV268-miR- 140 complexes were transiently transfected into cells of groups B and C. After transfection, real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expressions of exogenous miR-140; Annexin Ⅴ-FITC/PI double staining and MTT assay were used to detect the effect of exogenous miR-140 on apoptosis and proliferation of transfected chondrocytes; the expressions of Sox9, Aggrecan, and histone deacetylase 4 (Hdac4) were detected by RT-qPCR. RT-qPCR showed that the expression of miR-140 in group C was significantly higher than that in groups A and B ( P<0.05). Compared with groups A and B, the apoptosis rate in group C was decreased and the proliferation activity was improved, Sox9 and Aggrecan gene expressions were significantly up-regulated, and Hdac4 gene expression was significantly down-regulated ( P<0.05). There was no significant difference in above indexes between groups A and B ( P>0.05). Exogenous gene can be carried into the chondrocytes by NNSCS and expressed efficiently, the high expression of miR-140 can improve the biological activity of chondrocytes cultured in vitro, which provides important experimental basis for the treatment of cartilage damage diseases.

A Novel Method to Induce Cartilage Regeneration with Cubic Microcartilage

Cartilage tissue is characterized by its poor regenerative properties, and the clinical performance of cartilage grafts to replace cartilage defects has been unsatisfactory. Recently, cartilage regeneration with mature chondrocytes and stem cells has been developed and applied in clinical settings. However, there are challenges with the use of mature chondrocytes and stem cells for tissue regeneration, including the high costs associated with the standard stem cell isolation methods and the decreased cell viability due to cell manipulation. Previous studies demonstrated that cartilage can be regenerated from chondrocyte clusters that contain stem cells. Based upon some of the existing techniques, the goal of this study was to develop a novel and practical method to induce cartilage regeneration. A microslicer device was developed to process cartilage tissues into micron-size cartilage (microcartilage) in a minimally invasive manner. We evaluated microcartilage sizes and demonstrated 100-400 µm as optimal for generating a high cell yield with collagenase digestion. In addition, autologous intrafascial implantation of the composites of microcartilage and an absorbable scaffold with a slow-release system of basic fibroblast growth factor (bFGF) was carried out to induce cartilage regeneration. Our results demonstrated that the extent of bFGF diffusion depends on the size of microcartilage, and that cartilage regeneration was induced most effectively with 100 µm of microcartilage via SOX5 upregulation. These findings suggest that cartilage regeneration is possible with microcartilage as a source of cells without ex vivo cell expansion.

Application of a novel tyramine substituted hyaluronan gel for culture of mouse pre-antral follicles

Follicle culture in matrices that allow 3-D growth may better mimic in vivo conditions by preserving follicle architecture and critical granulosa-oocyte interactions.This report describes the application of a new biomaterial for in vitro follicle maturation (IVM) and the production of functionally competent oocytes. Mouse pre-antral follicle model for IVM and testing of a hylaluronan matrix. Ovaries from 12 -14 day SJLs pups were removed. Pre-antral follicles were collected by either mechanical dissection (M) or with collagenase (CL) digestion. Pre-antral follicles with 2-3 layers of granulosa cells were collected. Crosslinking of hyaluronan (HA) gel was initiated and 5-7ul drops were pipetted in to wells. Follicles were added before gelling was completed. Culture medium (25ul) was added to each well and along with an oil overlay. Follicles were cultured at 37C with 6% CO2 culture in α-MEM supplemented with 5% FBS, 100 mIU/ml FSH,10 mIU/ml LH and ITS. Final maturation was triggered with hCG (1.5 iu/ml) and EGF (5 ng/ml) after 12 days of growth. Follicles were monitored daily and medium was collected for hormone assay. Mature oocytes were inseminated with sperm and fertilization was assessed the following morning. Outcome parameters monitored were follicle morphology, survival to the end of the in vitro culture (IVC) interval, estradiol secretion,germinal vesicle breakdown, oocyte maturation to the metaphase II stage and 2-cell zygote formation following IVF insemination. Differences between treatments were analyzed using the Chi square and student T test. P value of <0.05 was considered significant. Average diameter of collagenase isolated follicles was 120.3 ± 5.4, with an average egg diameter of 61.1μm ± 2.1. Post maturation oocyte measurements for the non-HA and HA follicles were 87.9μm ± 4.4 and 86.2μm ± 6.4, respectively. Maturation rates for CL isolated follicles embedded in HA gels (75.7%) or cultured in medium alone(84.7%) were similar. A significantly lower maturation rate (59%) was obtained with mechanically isolated follicles embedded in HA gel.Therefore CL was used for subsequent experiments.Tabled 1IVM Summary DataHA GelHA GelNon-HA Gel(M)(CL)(CL)Number of Follicles Cultured122283293Ovulated COCs70144184Mature Oocytes41108155Maturation Rate (%)59 ± 0.7*75.7 ± 13.3284.7 ± 8.1Fertilization Rate (%)-88.7 ± 5.983.3 ± 10.1* Significantly different from CL. Open table in a new tab * Significantly different from CL. The HA matrix allowed continued follicle growth, oocyte maturation and resumption of meiosis. Functional competence of oocytes from HA matured follicles was confirmed by their ability to fertilize.Further testing and live birth data is needed to validate this culture model. HA is a natural matrix for cell growth and may prove especially advantageous when applied to follicles in large animal models requiring maintenance of follicular architecture over longer culture intervals.

Construction of tissue-engineered skin by culture of human amniotic epithelial cells on human de-epidermized dermis for the repair of skin defects in nude mice: an experimental study

Objective To evaluate the feasibility of repair of full-thickness skin defects in nude mice with tissue-engineered skin which was constructed by culture of human amniotic epithelial cells (hAECs) and fibroblasts on human de-epidermized dermis (DED) . Methods Healthy human amniotic tissues were treated with trypsin at a low concentration in multi-steps to prepare hAECs, and a two-step collagenase digestion was used to treat healthy children′s prepuce tissues to prepare fibroblast suspensions. When fibroblasts were cultured in vitro up to passage 3-5 and hAECs up to passage 2, they were seeded on the reticular dermal surface and basement membrane surface of the DED respectively to construct the tissue-engineered skin. A total of 20 healthy male nude mice aged 3-4 weeks were enrolled into this experiment, and full-thickness skin defects were made on the middle of the back of mice. Then, these mice were randomly divided into 2 groups by using a lottery method, and reconstructed full-thickness tissue-engineered skin grafts and vaseline oil gauze were used to cover the wounds in the tissue-engineered skin group and control group respectively. The whole body and transplantation sites of the nude mice were observed on day 7, 14, 21 and 28 after transplantation, the wound healing time and rate were compared between the above two groups, and skin tissues at the transplantation site were harvested at 4 weeks after transplantation and subjected to histological examination. Results HAECs had stem-cell characteristics and expressed octamer-binding protein-4 (OCT-4) and embryonic marker stage-specific embryonic antigen-4 (SSEA-4) . After 2-week organ culture, the in vitro reconstructed tissue-engineered skin showed 4-9 continuous layers of stratified epithelium, and the histological structure of the epidermis was similar to that of the normal human skin. Compared with the control group, the tissue-engineered skin group showed significantly higher wound healing rates on day 7, 14 and 21 after transplantation (57.49% ± 6.11% vs. 22.93% ± 4.26%, 92.80% ± 3.10% vs. 54.57% ± 7.94%, 98.83% ± 0.25% vs. 91.16% ± 4.79%, respectively; n= 10, t= 27.36, 32.23, 11.80, respectively, all P < 0.001) , shorter wound healing time[ (21.51 ± 1.51) d vs. (28.80 ± 1.14) d, n= 10, t= 42.23, P < 0.001], with the color of skin grafts closer to that of autologous skin on day 28 after transplantation. Histological examination revealed distinct stratification of the epithelium, obvious keratinization and favorable growth of cells in the dermis in the tissue-engineered skin group, but thin epithelium with some defects, indistinct stratification of the dermis, and inflammatory cell infiltration in the control group. Conclusion Tissue-engineered skin constructed by the culture of hAECs and fibroblasts on human DED can survive in nude mice after transplantation, resulting in a more favorable healing of wounds, and is expected to serve as a kind of ideal tissue-engineered skin. Key words: Tissue engineering; Epithelial cells; Dermis; Skin transplantation; Wound healing; De-epidermized dermis; Human amniotic epithelial cells

Adipose-derived stem cells: Comparison between two methods of isolation for clinical applications.

BackgroundAdipose-derived stem cells are recognized as being an effective mesenchymal stem cell population with enormous potential in different fields of regenerative medicine and stem cell therapy. Although there is unanimous agreement on the harvesting procedure for adipose tissue, there are various protocols for adipose-derived stem cell isolation. The aim of this study was compare two methods of adipose-derived stem cells (ASCs) isolation, one based on a mechanical + enzymatic (ME) procedure and the other one exclusively mechanical (MC), in order to determine which one was superior to the other in accordance with current European and US legislation.MethodsWe reported step by step the two different methods ASCs isolation by comparing them. The ME procedure included the use of a centrifuge, an incubator and collagenase digestion solution (Collagenase NB 6 GMP Grade 17458; Serva GmbH, Heidelberg, Germany). The MC procedure was performed by vibrating shaker and centrifuge, both placed in a laminar airflow bench.ResultsWith the ME procedure, a mean of 9.06 × 105 ASCs (range, 8.4 to 9.72 × 105; SD ± 6.6 × 105) was collected, corresponding to 25.9% of the total number of harvested cells. With the MC procedure, a mean of 5 × 105 ASCs (range: 4.0 to 6.0 × 105; SD, ±1 × 105) was collected, corresponding to 5% of the total number of harvested cells.ConclusionBased on data collected, from the same amount of lipoaspirate the ME procedure allowed to isolate a greater number of ASCs (25,9%) compared to the MC one (5%).

Imaging and modeling of acute pressure-induced changes of collagen and elastin microarchitectures in pig and human resistance arteries.

The impact of disease-related changes in the extracellular matrix (ECM) on the mechanical properties of human resistance arteries largely remains to be established. Resistance arteries from both pig and human parietal pericardium (PRA) display a different ECM microarchitecture compared with frequently used rodent mesenteric arteries. We hypothesized that the biaxial mechanics of PRA mirror pressure-induced changes in the ECM microarchitecture. This was tested using isolated pig PRA as a model system, integrating vital imaging, pressure myography, and mathematical modeling. Collagenase and elastase digestions were applied to evaluate the load-bearing roles of collagen and elastin, respectively. The incremental elastic modulus linearly related to the straightness of adventitial collagen fibers circumferentially and longitudinally (both R2 ≥ 0.99), whereas there was a nonlinear relationship to the internal elastic lamina elastin fiber branching angles. Mathematical modeling suggested a collagen recruitment strain (means ± SE) of 1.1 ± 0.2 circumferentially and 0.20 ± 0.01 longitudinally, corresponding to a pressure of ~40 mmHg, a finding supported by the vital imaging. The integrated method was tested on human PRA to confirm its validity. These showed limited circumferential distensibility and elongation and a collagen recruitment strain of 0.8 ± 0.1 circumferentially and 0.06 ± 0.02 longitudinally, reached at a distending pressure below 20 mmHg. This was confirmed by vital imaging showing negligible microarchitectural changes of elastin and collagen upon pressurization. In conclusion, we show here, for the first time in resistance arteries, a quantitative relationship between pressure-induced changes in the extracellular matrix and the arterial wall mechanics. The strength of the integrated methods invites for future detailed studies of microvascular pathologies.NEW & NOTEWORTHY This is the first study to quantitatively relate pressure-induced microstructural changes in resistance arteries to the mechanics of their wall. Principal findings using a pig model system were confirmed in human arteries. The combined methods provide a strong tool for future hypothesis-driven studies of microvascular pathologies.

Abstract 3972: Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids

Abstract Despite the impressive activity of PD-1/PD-L1 immune checkpoint blockade in melanoma, lung, and other cancers, the majority of patients are intrinsically resistant to this therapy. Furthermore, despite years of effort few robust biomarkers of response exist, and tumor PD-L1 expression and mutational load are only partially predictive of activity. Nearly all attempts at biomarker development have relied on fixed tumor tissue or studies in plasma. Meanwhile, emerging functional approaches to study patient derived samples, such as organoid development and circulating tumor cell culture, lack features of the native tumor immune microenvironment and thus are limited in the ability to assess ex vivo anti-PD-1 response. We have developed a novel approach to study short-term ex vivo response to immune checkpoint blockade based upon short term culture of murine- and patient-derived organotypic tumor spheroids (MDOTS/PDOTS) in a 3-dimensional microfluidic system. Spheroids derived from fresh mouse and human tumor samples through partial collagenase digestion and filtration retain autologous tumor infiltrating immune cells, including PD-1 positive CD8 T lymphocytes. Using murine syngeneic mouse cancer models with known responsiveness or resistance to PD-1 blockade, we demonstrate the ability to recapitulate tumor killing ex vivo or lack thereof. Multiplexed cytokine profiling of conditioned media in this system further enables characterization of the cytokine and chemokine response to PD-1 and/or CTLA-4 blockade. We thus analyzed the secreted cytokine/chemokine response to PD-1 inhibition across a large panel of PDOTS, and observed pronounced induction of CCL19/CXCL13, which associated with % PD-1 positive CD8 T cells. This strategy further uncovered production of a class of myeloid attracting chemokines and cytokines in major proportion of samples, which clustered with the presence of plasmacytoid dendritic cells and myeloid derived suppressor cells. Importantly, the presence of this maladaptive cytokine/chemokine signature correlated with lack of response in the subset of patients that had been treated with PD-1 blockade. Together, these data demonstrate the ability to profile the functional response to tumor PD-1 blockade ex vivo and unveil a novel strategy to advance precision immuno-oncology. Citation Format: David A. Barbie, Russell Jenkins, Kwok-Kin Wong. Ex vivo profiling of PD-1 blockade using organotypic tumor spheroids [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3972. doi:10.1158/1538-7445.AM2017-3972

Battling bacterial infection with hexamethylene diisocyanate cross-linked and Cefaclor-loaded collagen scaffolds

Implant infections remain a major healthcare problem due to the prolonged hospitalisation period required to disrupt and treat bacterial biofilm formation, and the need for additional surgery to remove/replace the infected implant, which if not removed in a timely manner may lead to sepsis. Although localised drug administration, via an implanted scaffold, has shown promise in a clinical setting, the ideal scaffold cross-linking (to initially withstand the aggressive infection environment) and drug (to be effective against infection) have yet to be identified. In this work, in the first instance, the biochemical, biophysical, and biological properties of collagen sponges as a function of various concentrations (0.625%, 1.0%, 2.5%, 5.0%, and 10.0%) of hexamethylene diisocyanate were assessed. Data presented illustrate that hexamethylene diisocyanate at 0.625% concentration was able to effectively stabilise collagen scaffolds, as judged by the reduction in free amines, adequate resistance to collagenase digestion, reduction in swelling, increase in denaturation temperature, suitable mechanical properties, and appropriate cytocompatibility. Subsequently, collagen scaffolds stabilised with 0.625% hexamethylene diisocyanate were loaded with variable concentrations (0, 10, 100, and 500 μg ml−1) of Cefaclor and Ranalexin. Both drugs exhibited similar loading efficiency, release profile, and cytocompatibility. However, only collagen scaffolds loaded with 100 μg ml−1 Cefaclor exhibited adequate antibacterial properties against both 106 and 108 colony-forming units per ml of both Escherichia coli and Staphylococcus epidermidis.

Angle-ply biomaterial scaffold for annulus fibrosus repair replicates native tissue mechanical properties, restores spinal kinematics, and supports cell viability.

The quality of life for millions of individuals globally is detrimentally impacted by IVD degeneration and herniation. These pathologies often result in the structural demise of IVD tissue, particularly the annulus fibrosus (AF). Biomaterials developed for AF repair have yet to demonstrate the mechanical strength and durability required for utilization in the spine. Herein, we demonstrate the development of an angle-ply AF repair patch (AFRP) that can resist the application of physiologically relevant stresses without failure and which contributes to the restoration of functional spinal unit axial kinematics following repair. Furthermore, we show that this biomaterial can resist accelerated degradation in a simulated degenerate environment and supports AF cell viability.

Effects of Collagenase Digestion and Stromal Vascular Fraction Supplementation on Volume Retention of Fat Grafts.

The use of autologous fat as a soft tissue filler has increased over the past decade in both reconstructive and aesthetic surgeries. Enhancement of autologous fat grafts with the addition of the stromal vascular fraction (SVF) has been reported to improve long-term volume retention. Stromal vascular fraction is most commonly isolated using enzymatic digestion, but it is unknown what effect the digestion process has on the adipocytes and SVF cells that comprise the graft. Some clinicians have reported use of enzymatically digested fat grafts to alter the physical properties of the tissue in specialized applications. We have previously reported that increasing collagenase digestion duration adversely affects the viability of adipocytes and SVF cells. Here, we aimed to determine if collagenase digestion of adipocytes before grafting is detrimental to long-term graft retention and if SVF supplementation can abrogate these potential deleterious effects. We used a published xenograft model in which human lipoaspirate was implanted into the scalp of immunocompromised mice to study the effects of collagenase digestion on in vivo graft survival after 12 weeks. We used 4 experimental groups: grafts composed of collagenase-digested and nondigested adipocytes (50-minute digestion) and grafts with and without SVF supplementation. We used microcomputed tomography to serially and noninvasively quantify graft volume, in conjunction with hematoxylin-eosin staining of histological cross-sections of implanted and excised grafts to assess overall tissue viability. We found that adipocytes that were collagenase-digested before implantation had significantly lower retention rates at 12 weeks and poorer tissue health, which was assessed by quantifying the number of intact adipocytes, the number of cystic formations, and by scoring the degree of inflammation and fibrosis. Further, we found that SVF supplementation of the digested grafts improved graft survival, but not to the level observed in undigested grafts. We conclude that collagenase digestion adversely affects the long-term volume retention of fat grafts, but that graft retention is improved by SVF supplementation. These experimental results can serve as an initial framework to further elucidate the reported efficacy and safety of using collagenase-digested fat grafts and SVF in the clinical setting.

Effects of liver depression and psychological stress on human uterine leiomyoma cells by an AR–cAMP–PKA signal transduction pathway

ObjectiveBased on the emotional theory of Traditional Chinese Medicine, and combined with the modern medicine theory of psychological stress, a research model of human uterine leiomyoma cells (ULM) was cultured in vitro to determine the effectiveness of adrenergic receptor (AR) agonists in human ULM cell growth. In addition, we studied the functional influence of “liver depression and psychological stress theory” on fibroid formation by intervening in the AR–cAMP–PKA signaling pathway. The intention was to establish a new method to prevent and cure fibroids through “liver depression and psychological stress theory” and provide an experimental basis for the Traditional Chinese Medicine emotional theory. Materials and methodsPrimary human ULM cells were enriched by collagenase digestion. Immunohistochemistry and hematoxylin and eosin (HE) staining were used for cytological identification. Using this model, we studied intervention using specific AR agonists on ULM cells to observe the influence of “liver depression and psychological stress theory” on estrogen receptor (ER), progesterone receptor (PR), vascular endothelial growth factor (VEGF) and fibroblast growth factors (FGF). ResultsNorepinephrine (NE) and epinephrine (E) are adrenergic receptor agonists. They promoted ULM cell proliferation and increased the levels of ER, PR, VEGF and FGF. In contrast, isoproterenol (ISO) inhibited ULM cell proliferation and decreased the levels of ER, PR, VEGF and FGF. The protein expression of cAMP and PKA in ULM cells was reduced and the levels of ER, PR, VEGF and FGF were increased when co-treatment with the α-AR blocker (phentolamine). The β-AR blocker (metoprolol) displayed an opposite effect. ConclusionsAR agonists modulated ER, PR, VEGF and FGF levels in ULM cells in an AR–cAMP–PKA-dependent signaling pathways to influence fibroid occurrence and development.

The equine glucose-dependent insulinotropic polypeptide receptor: A potential therapeutic target for insulin dysregulation.

Metabolic disease is a significant problem that causes a range of species-specific comorbidities. Recently, a better understanding of glucose-dependent insulinotropic polypeptide (GIP) biology has led to the suggestion that inhibiting its action may attenuate obesity in several species. In horses, antagonism of GIP may also reduce hyperinsulinemia, which leads to insulin-associated laminitis, a painful comorbidity unique to this species. However, little is known about GIP in horses. The aims of this study were to examine the tissue distribution of equine GIP receptors (eGIPR), to determine whether eGIPR can be blocked using a GIP antagonist not tested previously in horses, and to establish whether there is any association between GIP concentrations and body mass in this species. Archived tissues from healthy horses were used to establish that eGIPR gene expression was strong in pancreas, heart, liver, kidney, and duodenum and absent in gluteal muscle. Pancreatic islets were isolated from fresh horse pancreas using collagenase digestion and layering through a density gradient. Islet viability was confirmed microscopically and by demonstrating that insulin production was stimulated by glucose in a concentration-dependent manner. Insulin release was also shown to be concentration-dependent with GIP up to 0.1µM, and the response to GIP was decreased ( = 0.037) by the antagonist (Pro3)GIP. As for the relationship between body mass and GIP in vivo postprandial GIP concentrations in archived plasma samples were positively correlated with body condition and cresty neck scores ( < 0.05). Thus, the eGIPR is a potential therapeutic target for insulin dysregulation and obesity in horses.

Exosomes from mesenchymal stem cells induce the conversion of hepatocytes into progenitor oval cells

BackgroundWe previously reported that mesenchymal stem cells (MSCs) possess therapeutic effects in a murine model of carbon tetrachloride-induced acute liver failure. In the study, we observed that the majority of repopulated hepatocytes were of recipient origin and were adjacent to transplanted MSCs; only a low percentage of repopulated hepatocytes were from transplanted MSCs. The findings indicate that MSCs guided the formation of new hepatocytes. Exosomes are important messengers for paracrine signaling delivery. The aim of this study is to investigate the paracrine effects, in particular, the effects of exosomes from MSCs, on hepatocytes.MethodsMature hepatocytes were isolated from murine liver by a two-step perfusion method with collagenase digestion. MSCs were obtained from murine bone marrow, and conditioned medium (CM) from MSC culture was then collected. Time-lapse imaging was used for observation of cell morphological change induced by CM on hepatocytes. In addition, expression of markers for hepatic progenitors including oval cells, intrahepatic stem cells, and hepatoblasts were analyzed.ResultsTreatment with the CM promoted the formation of small oval cells from hepatocytes; time-lapse imaging demonstrated the change from epithelial to oval cell morphology at the single hepatocyte level. Additionally, expression of EpCAM and OC2, markers of hepatic oval cells, was upregulated. Also, the number of EpCAMhigh cells was increased after CM treatment. The EpCAMhigh small oval cells possessed colony-formation ability; they also expressed cytokeratin 18 and were able to store glycogen upon induction of hepatic differentiation. Furthermore, exosomes from MSC-CM could induce the conversion of mature hepatocytes to EpCAMhigh small oval cells.ConclusionsIn summary, paracrine signaling through exosomes from MSCs induce the conversion of hepatocytes into hepatic oval cells, a mechanism of action which has not been reported regarding the therapeutic potentials of MSCs in liver regeneration. Exosomes from MSCs may therefore be used to treat liver diseases. Further studies are required for proof of concept of this approach.

In vitrodifferentiation of human amniotic mesenchymal stem cells into ligament fibroblasts after induced by transforming growth factor β 1 and vascular endothelial growth factor

To investigate whether human amniotic mesenchymal stem cells (hAMSCs) have the characteristics of mesenchymal stem cells (MSCs) and the differentiation capacity into ligament fibroblasts in vitro. The hAMSCs were separated through trypsin and collagenase digestion from placenta, the phenotypic characteristics of hAMSCs were detected by flow cytometry, the cytokeratin-19 (CK-19) and vimentin expression of hAMSCs were tested through immunofluorescence staining. The hAMSCs at the 3rd passage were cultured with L-DMEM/F12 medium containing transforming growth factor β 1 (TGF-β 1) and vascular endothelial growth factor (VEGF) as the experimental group and with single L-DMEM/F12 medium as the control group. The morphology of hAMSCs was observed by inverted phase contrast microscope; the cellular activities and ability of proliferation were examined by cell counting kit-8 (CCK-8) method; the ligament fibroblasts related protein expressions including collagen type I, collagen type III, Fibronectin, and Tenascin-C were detected by immunofluorescence staining; specific mRNA expressions of ligament fibroblasts and angiogenesis including collagen type I, collagen type III, Fibronectin, α-smooth muscle actin (α-SMA), and VEGF were measured by real-time fluorescence quantitative PCR. The hAMSCs presented monolayer and adherent growth under inverted phase contrast microscope; the flow cytometry results demonstrated that hAMSCs expressed the MSCs phenotypes; the immunofluorescence staining results indicated the hAMSCs had high expression of the vimentin and low expression of CK-19; the hAMSCs possessed the differentiation ability into the osteoblasts, chondroblasts, and lipoblasts. The CCK-8 results displayed that cells reached the peak of growth curve at 7 days in each group, and the proliferation ability in the experimental group was significantly higher than that in the control group at 7 days ( P<0.05). The immunofluorescence staining results showed that the expressions of collagen type I, collagen type III, Fibronectin, and Tenascin-C in the experimental group were significantly higher than those in the control group at 5, 10, and15 days after culture ( P<0.05). The real-time fluorescence quantitative PCR results revealed that the mRNA relative expressions had an increasing tendency at varying degrees with time in the experimental group ( P<0.05). The relative mRNA expressions of collagen type I, collagen type III, Fibronectin, α-SMA, and VEGF in the experimental group were significantly higher than those in the control group at the other time points ( P<0.05), but no significant difference was found in the relative mRNA expressions of collagen type I, collagen type III, and VEGF between 2 groups at 5 days ( P>0.05). The hAMSCs possesses the characteristics of MSCs and good proliferation ability which could be chosen as seed cell source in tissue engineering. The expressions of ligament fibroblasts and angiogenesis related genes could be up-regulated, after induction in vitro, and the synthesis of ligament fibroblasts related proteins could be strengthened. In addition, the application of TGF-β 1 and VEGF could be used as growth factors sources in constructing tissue engineered ligament.

Sheep Placenta Cotyledons: A Noninvasive Source of Ovine Mesenchymal Stem Cells.

Sheep are one of the most frequently used large animal models in stem cell research. However, minimal invasive or noninvasive sources of mesenchymal stem cells (MSCs) in sheep are scarce. In the light of the principles of the 3Rs (reduce, refine, replace), it would therefore be desirable to identify a minimally invasive or noninvasive ovine MSC source. In humans, the chorionic villi of the placenta, which can be noninvasively harvested as part of the afterbirth, have been identified as a rich source of MSCs. Therefore, in the present study, ovine placenta cotyledons, which have similar function and structure to human chorionic villi, were tested for their potential use as a noninvasive source of ovine MSCs. Through mincing of the placental cotyledons, collagenase digestion, and Ficoll density gradient centrifugation, combined with plastic adherence selection, MSCs were successfully isolated. Their morphological, immunophenotypical, and cellular growth characteristics, as well as their proliferation, differentiation, and migration potential, were evaluated and compared to the currently best-researched MSC source, bone marrow-derived stem cells. Ovine cotyledons were shown to be a reliable, abundant source for the noninvasive, pain- and risk-free harvest of MSCs. The collection procedure does not interfere with partum or the initial bonding phase between ewes and lambs and is therefore exempt from ethical debate. Ovine placenta cotyledon-derived MSCs exhibit multipotential characteristics and can be cryopreserved for later use.