Shuyu Piao

Therapeutic lymphangiogenesis using stem cell and VEGF-C hydrogel

Lymphedema is a manifestation of lymphatic system insufficiency. It arises from primary lymphatic dysplasia or secondary obliteration after lymph node dissection or irradiation. Although improvement of swelling can be achieved by comprehensive non-operative therapy, treatment of this condition requires lifelong care and good compliance. Recently molecular-based treatments using VEGF-C have been investigated by several researchers. We designed the present study to determine whether the therapeutic efficacy of implanted human adipose-derived stem cells (hADSCs) could be improved by applying a gelatin hydrogel containing VEGF-C (VEGF-C hydrogel) to the site of tissue injury in a lymphedema mouse model. Four weeks after the operation, we evaluated edema and determined lymphatic vessel density at various post-operative time points. Mice treated with hADSCs and VEGF-C hydrogel showed a significantly decreased dermal edema depth compared to the groups of mice that received hADSCs only or VEGF-C hydrogel only. Immunohistochemical analysis also revealed that the hADSC/VEGF-C hydrogel group showed significantly greater lymphatic vessel regeneration than all the other groups. hADSCs were detected in the implantation sites of all mice in the hADSC/VEGF-C group, and exhibited a lymphatic endothelial differentiation phenotype as determined by co-staining PKH-labeled hADSCs for the lymphatic marker LYVE-1. Our results suggest that co-administration of hADSCs and VEGF-C hydrogel has a substantial positive effect on lymphangiogenesis.

Combination therapy of human adipose-derived stem cells and basic fibroblast growth factor hydrogel in muscle regeneration

Skeletal muscle regeneration after sport injury is inconsistent, and complete healing without fibrosis is very important. In this study, we determined whether the combination therapy using human adipose-derived stem cells (h-ADSCs) and basic fibroblast growth factor (bFGF) incorporated into hydrogel could enhance muscle regeneration in a muscle laceration animal model. The h-ADSCs and/or bFGF hydrogels were applied to the lacerated gastrocnemius muscle. Fast twitch muscle contraction improved significantly and fibrosis decreased significantly in combined h-ADSC and bFGF-hydrogel group compared to other experimental groups. Skeletal muscle differentiation of h-ADSCs was determined by immunohistochemistry (PKH-26/MyHC co-staining) and Western blot. Our data suggested that combination therapy of h-ADSCs and bFGF hydrogel resulted in functional recovery, revascularization and reinnervation with minimal fibrosis in lacerated muscle. A combination of h-ADSCs and bFGF hydrogel can be used as a promising therapy for skeletal muscle regeneration.

Combined therapeutic effect of udenafil and adipose-derived stem cell (ADSC)/brain-derived neurotrophic factor (BDNF)-membrane system in a rat model of cavernous nerve injury.

OBJECTIVE To prevent cavernous nerve injury and corpus cavernosum apoptosis-induced erectile dysfunction (ED) after prostatectomy surgery, we investigated whether oral administration of udenafil combination with covering adipose-derived stem cells (ADSCs) and brain-derived neurotrophic factor (BDNF) immobilized poly-lactic-co-glycolic (PLGA) membrane on the injured cavernous nerve could further improve erectile dysfunction. METHODS Adult Sprague-Dawley rats were divided into 5 groups: normal group (sham-operated group), bilateral cavernous nerve injury (BCNI) group (BCNI group), udenafil group (oral administration of udenafil 20 mg/kg daily), AB group (BCNI group with ADSCs covered with BDNF membrane on cavernous nerve), AB/udenafil group (AB group with udenafil group). After 4 weeks, erectile function was examined before tissue harvest. Penile tissues were evaluated in terms of the expression of smooth muscle actin (SMA), neuronal nitric oxide synthase (nNOS), and vascular endothelial growth factor (VEGF). The cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum was quantified by cGMP assay. RESULTS AB/udenafil treatment markedly improved erectile function and prevented the architecture damage of the corpus cavernosum, compared with other treated groups. Udenafil had no statistical significance on increasing nNOS expression, but enhanced VEGF expression. On the contrary, the AB group had no statistical significance on enhancing VEGF expression, but increased nNOS expression. AB/udenafil treatment significantly increased nNOS expression, VEGF expression, and elevated cGMP level, compared with the udenafil group and AB group. CONCLUSION The orally administered udenafil combination with ADSC/BDNF-membrane system protected cavernous nerve and improved angiogenesis in the corpus cavernosum, which further maintained erectile function in a rat model of postprostatectomy erectile dysfunction.

Therapeutic Effect of Adipose-Derived Stem Cells and BDNF-immobilized PLGA Membrane in a Rat Model of Cavernous Nerve Injury

INTRODUCTION Cavernous nerve injury is the main reason for post-prostatectomy erectile dysfunction (ED). Stem cell and neuroprotection therapy are promising therapeutic strategy for ED. AIM To evaluate the therapeutic efficacy of adipose-derived stem cells (ADSCs) and brain-derived neurotrophic factor (BDNF) immobilized Poly-Lactic-Co-Glycolic (PLGA) membrane on the cavernous nerve in a rat model of post-prostatectomy ED. Methods.  Rats were randomly divided into five groups: normal group, bilateral cavernous nerve crush injury (BCNI) group, ADSC (BCNI group with ADSCs on cavernous nerve) group, BDNF-membrane (BCNI group with BDNF/PLGA membrane on cavernous nerve) group, and ADSC/BDNF-membrane (BCNI group with ADSCs covered with BDNF/PLGA membrane on cavernous nerve) group. BDNF was controlled-released for a period of 4 weeks in a BDNF/PLGA porous membrane system. MAIN OUTCOME MEASURES Four weeks after the operation, erectile function was assessed by detecting the ratio of intra-cavernous pressure (ICP)/mean arterial pressure (MAP). Smooth muscle and collagen content were determined by Massons trichrome staining. Neuronal nitric oxide synthase (nNOS) expression in the dorsal penile nerve was detected by immunostaining. Phospho-endothelial nitric oxide synthase (eNOS) protein expression and cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum were quantified by Western blotting and cGMP assay, respectively. RESULTS In the ADSC/BDNF-membrane group, erectile function was significantly elevated, compared with the BCNI and other treated groups. ADSC/BDNF-membrane treatment significantly increased smooth muscle/collagen ratio, nNOS content, phospho-eNOS protein expression, and cGMP level, compared with the BCNI and other treated groups. CONCLUSIONS ADSCs with BDNF-membrane on the cavernous nerve can improve erectile function in a rat model of post-prostatectomy ED, which may be used as a novel therapy for post-prostatectomy ED.

Comparison of human muscle-derived stem cells and human adipose-derived stem cells in neurogenic trans-differentiation.

Purpose Erectile dysfunction (ED) remains a major complication from cavernous nerve injury during radical prostatectomy. Recently, stem cell treatment for ED has been widely reported. This study was conducted to investigate the availability, differentiation into functional cells, and potential of human muscle-derived stem cells (hMDSCs) and human adipose-derived stem cells (hADSCs) for ED treatment. Materials and Methods We compared the neural differentiation of hMDSCs and hADSCs. Human muscle and adipose tissues were digested with collagenase, followed by filtering and centrifugation. For neural induction, isolated hMDSCs and hADSCs were incubated in neurobasal media containing forskolin, laminin, basic-fibroblast growth factor, and epidermal growth factor for 5 days. Following neural induction, hMDSCs and hADSCs were differentiated into neural cells, including neurons and glia, in vitro. Results In neural differentiated hMDSCs (d-hMDSCs) and differentiated hADSCs (d-hADSCs), neural stem cell marker (nestin) showed a significant decrease by immunocytochemistry, and neuronal marker (β-tubulin III) and glial marker (GFAP) showed a significant increase, compared with primary hMDSCs and hADSCs. Real-time chain reaction analysis and Western blotting demonstrated significantly elevated levels of mRNA and protein of β-tubulin III and GFAP in d-hADSCs compared with d-hMDSCs. Conclusions We demonstrated that hMDSCs and hADSCs can be induced to undergo phenotypic and molecular changes consistent with neurons. The neural differentiation capacity of hADSCs was better than that of hMDSCs.

Comparison Between Subcutaneous Injection of Basic Fibroblast Growth Factor-Hydrogel and Intracavernous Injection of Adipose-derived Stem Cells in a Rat Model of Cavernous Nerve Injury

OBJECTIVE To compare the effects of subcutaneous penile injection of basic fibroblast growth factor (bFGF)-hydrogel and intracavernous injection of human adipose-derived stem cells (h-ADSCs) on improving erectile function in a rat model of cavernous nerve injury. MATERIALS AND METHODS Adult male Sprague-Dawley rats were randomly divided into 5 groups (n = 10 per group): age-matched control (normal group), bilateral cavernous nerve injury (BCNI group), penile subcutaneous injection of hydrogel after BCNI (hydrogel group), penile subcutaneous injection of bFGF-hydrogel after BCNI (bFGF-hydrogel group) and intracavernous injection of h-ADSCs after BCNI (ADSC group). Four weeks after the treatment, all rats underwent an erectile function test. Then, penile tissue was harvested for immunohistological analysis of bFGF, phalloidin, and cluster of differentiation (CD) 31. The cyclic guanosine monophosphate (cGMP) level of the corpus cavernosum was quantified by cGMP assay. RESULTS From the functional test and immunohistological result, we observed that bFGF-hydrogel and h-ADSCs injection significantly elevated intracavernous pressure. The evaluation of filamentous actin content, CD31 expression, and cGMP concentration in the corpus cavernosum were meaningfully increased in the bFGF-hydrogel and ADSC groups compared with BCNI group. The bFGF released from bFGF-hydrogel prevented smooth muscle atrophy. Moreover, bFGF expression was significantly increased in bFGF-hydrogel group. CONCLUSION The subcutaneous injection of bFGF-hydrogel prevented smooth muscle atrophy, increased the intracavernous pressure, and improved erectile function like an intracavernous injection of h-ADSCs.

Comparison of three types of stress urinary incontinence rat models: electrocauterization, pudendal denervation, and vaginal distension.

OBJECTIVE To investigate the differences in the histopathologic and functional characteristics of 3 rat models of stress urinary incontinence. MATERIALS AND METHODS A total of 24 female, 10-week-old, Sprague-Dawley rats were randomly divided into 4 groups: normal, electrocauterization, pudendal denervation, and vaginal distension. At 2 weeks after surgery, the leak point pressure was measured to detect urinary leakage. Urethral tissue samples were collected for histological examination. RESULTS The smooth muscle content in the electrocauterization group was significantly decreased compared with that in all other groups, indicating that electrocauterization caused the most severe injury. A blood vessel marker, von Willebrand factor, was co-stained with α-smooth muscle actin to detect the blood vessel distribution. No significant differences were seen in von Willebrand factor expression among the 4 groups, other than in the electrocauterization group, in which we could hardly observe blood vessel expression. Protein gene product 9.5 staining was used to detect nerve fibers and cells. Protein gene product 9.5 expression was significantly lower in all the treatment groups compared with that in the normal group (P <.05), in particular, in the electrocauterization and pudendal denervation groups (P <.01). The leak point pressure was significantly lower in the electrocauterization (P <.01), pudendal denervation (P <.01), and vaginal distension (P <.05) groups than in the normal group. CONCLUSION The vaginal distension model should mainly be used as the myogenic damage stress urinary incontinence animal model; the pudendal denervation model mainly as the neurogenic damage stress urinary incontinence animal model; and the electrocauterization model as the vasculogenic, neurogenic, and myogenic damage animal model.

Effect of Human Muscle–derived Stem Cells on Cryoinjured Mouse Bladder Contractility

OBJECTIVE To investigate the effect of human muscle-derived stem cells (hMDSCs) on ameliorating impaired detrusor contractility in a cryoinjured bladder murine model. METHODS The hMDSCs were isolated and cultured by modified preplate technique, and only CD34-positive hMDSCs were extracted by Mini-MACS kits. Isolated hMDSCs were prelabeled with PKH26 and injected into the cryoinjured bladder to observe the pattern and characteristics. The nude mice were subdivided into three groups: normal group (N), cryoinjury bladder group with saline injection (C), and hMDSCs injection group after cryoinjury (M). At 2 weeks after injecting hMDSCs, we compared the contractility of bladder muscle strip stimulated by electrical field stimulation (EFS), acetylcholine (Ach.), and adenosine triphosphate (ATP), and the bladder smooth muscle tissue was examined by immunohistochemistry. RESULTS The contractile powers of bladder muscle strip in the C group were more decreased than the N group after EFS, Ach, and ATP treatment (P < .05). The bladder contractility of the M group was more increased than in the C group (P < .05), but was lower than the N group after EFS and Ach treatment. However, there was no significant difference of contractile power between the C and M groups after ATP stimulation. In immunohistochemical staining, the thickness of the bladder smooth muscle layer in the M group was significantly increased compared with the C group, and PKH26-labeled implanted cells were positive for smooth muscle cell differentiation marker (α-SMA) in the injected region. CONCLUSION hMDSCs injection increased cholinergic bladder contractile power but not the purinergic component of bladder contraction after cryoinjury.

The Effect of PnTx2-6 Protein From Phoneutria nigriventer Spider Toxin on Improvement of Erectile Dysfunction in a Rat Model of Cavernous Nerve Injury

OBJECTIVE To explore the therapeutic potential of PnTx2-6 injected 3 times a week for 4 weeks into the intracavernosal tissue in a rat model of bilateral cavernous nerve crush injury (BCNI). METHODS Eight-week-old male Sprague-Dawley rats were randomly divided into the following 6 groups (n = 5 per group): age-matched control (normal group), BCNI (injury group), post-BCNI phosphate-buffered saline injection (PBS group), post-BCNI Sf9 cell-lysate injection (N/C group), post-BCNI injection of cell lysate from S9 cells infected with wild-type recombinant baculovirus (W/T group), and post-BCNI injection of cell lysate from S9 cells infected with recombinant baculovirus containing PnTx2-6 (PnTx2-6 group). Injections were delivered 3 times a week for 4 weeks. After 4 weeks, intracavernosal pressure-to-mean arterial pressure ratio, smooth muscle and collagen content via the Masson trichrome staining, levels of neural nitric oxide synthase, phosphoendothelial nitric oxide synthase, and cyclic guanosine monophosphate were all measured. RESULTS The PnTx2-6 group showed significantly higher intracavernosal pressure-to-mean arterial pressure ratio (P <.05), smooth muscle-to-collagen ratio (P <.01), expression levels of neural nitric oxide synthase, phosphoendothelial nitric oxide synthase (P <.05), and cyclic guanosine monophosphate (P <.05) than all other experimental groups. CONCLUSION We conclude that PnTx2-6 improved erectile function and prevented muscle atrophy in a rat model of BCNI via increased synthesis of nitric oxide and cyclic guanosine monophosphate.

The effect of a bioactive tissue-engineered sling in a rat of stress incontinence model.

In this study, we attempt to examine the feasibility of the bioactive tissue-engineered sling by using muscle precursor cells (MPCs)-seeded Poly(ε-caprolactone) (PCL) nanofiber sheet in a rat model of stress urinary incontinence (SUI). In vitro, MPCs were cultured on a PCL nanofiber sheet for one week, where the MPCs-seeded PCL nanofiber sheet showed constant twitching contraction by electrical field stimulation in an organ bath. In vivo, MPCs-seeded PCL nanofiber sheet was placed under the female rats urethra after pudendal nerve denervation (animal model of SUI). The leak point pressure (LPP) was evaluated with the vertical tilt table after the operation for four weeks. The resulting LPP of MPCs-seeded PCL nanofiber sheet group was observed to be significantly higher than the denervation-only groups. Furthermore, PKH-26-labeled MPCs were observed under the urethral sphincter by immunohistochemistry. These results indicated that, the MPCs-seeded PCL nanofiber sheet have not only provided support for the deficient sphincter, but also actively improved the sphincters function overall. In conclusion, this bioactive tissue-engineered sling could be used as an ideal material for the treatment of SUI.