Microtissues Research Articles

Cellular self-assembly into 3D microtissues enhances the angiogenic activity and functional neovascularization capacity of human cardiopoietic stem cells.

While cell therapy has been proposed as next-generation therapy to treat the diseased heart, current strategies display only limited clinical efficacy. Besides the ongoing quest for the ideal cell type, in particular the very low retention rate of single-cell (SC) suspensions after delivery remains a major problem. To improve cellular retention, cellular self-assembly into 3D microtissues (MTs) prior to transplantation has emerged as an encouraging alternative. Importantly, 3D-MTs have also been reported to enhance the angiogenic activity and neovascularization potential of stem cells. Therefore, here using the chorioallantoic membrane (CAM) assay we comprehensively evaluate the impact of cell format (SCs versus 3D-MTs) on the angiogenic potential of human cardiopoietic stem cells, a promising second-generation cell type for cardiac repair. Biodegradable collagen scaffolds were seeded with human cardiopoietic stem cells, either as SCs or as 3D-MTs generated by using a modified hanging drop method. Thereafter, seeded scaffolds were placed on the CAM of living chicken embryos and analyzed for their perfusion capacity in vivo using magnetic resonance imaging assessment which was then linked to a longitudinal histomorphometric ex vivo analysis comprising blood vessel density and characteristics such as shape and size. Cellular self-assembly into 3D-MTs led to a significant increase of vessel density mainly driven by a higher number of neo-capillary formation. In contrast, SC-seeded scaffolds displayed a higher frequency of larger neo-vessels resulting in an overall 1.76-fold higher total vessel area (TVA). Importantly, despite that larger TVA in SC-seeded group, the mean perfusion capacity (MPC) was comparable between groups, therefore suggesting functional superiority together with an enhanced perfusion efficacy of the neo-vessels in 3D-MT-seeded scaffolds. This was further underlined by a 1.64-fold higher perfusion ratio when relating MPC to TVA. Our study shows that cellular self-assembly of human cardiopoietic stem cells into 3D-MTs substantially enhances their overall angiogenic potential and their functional neovascularization capacity. Hence, the concept of 3D-MTs may be considered to increase the therapeutic efficacy of future cell therapy concepts.

Wharton's jelly-derived mesenchymal cells as a new source for the generation of microtissues for tissue engineering applications.

Microtissues (MT) are currently considered as a promising alternative for the fabrication of natural, 3D biomimetic functional units for the construction of bio-artificial substitutes by tissue engineering (TE). The aim of this study was to evaluate the possibility of generating mesenchymal cell-based MT using human umbilical cord Wharton's jelly stromal cells (WJSC-MT). MT were generated using agarose microchips and evaluated ex vivo during 28days. Fibroblasts MT (FIB-MT) were used as control. Morphometry, cell viability and metabolism, MT-formation process and ECM synthesis were assessed by phase-contrast microscopy, functional biochemical assays, and histological analyses. Morphometry revealed a time-course compaction process in both MT, but WJSC-MT resulted to be larger than FIB-MT in all days analyzed. Cell viability and functionality evaluation demonstrated that both MT were composed by viable and metabolically active cells, especially the WJSC during 4-21days ex vivo. Histology showed that WJSC acquired a peripheral pattern and synthesized an extracellular matrix-rich core over the time, what differed from the homogeneous pattern observed in FIB-MT. This study demonstrates the possibility of using WJSC to create MT containing viable and functional cells and abundant extracellular matrix. We hypothesize that WJSC-MT could be a promising alternative in TE protocols. However, future cell differentiation and in vivo studies are still needed to demonstrate the potential usefulness of WJSC-MT in regenerative medicine.

Goat tendon collagen-human fibrin hydrogel for comprehensive parametric evaluation of HUVEC microtissue-based angiogenesis

The cell and extracellular matrix (ECM) interactions play a very important role during angiogenesis. Remodeling of the extracellular matrix along with pro-angiogenic/anti-angiogenic factors, and matrix-degrading proteases, accounts for endothelial cell growth, migration, and tube formation. However, for studying angiogenesis, only limited and expensive biomaterials are available. Despite being biocompatible, inexpensive, and easy availability; the potential of goat tendon collagen (GTC) has never been explored for vascular tissue engineering applications. Hence, the current investigation was focused on evaluating GTC as an alternative matrix for HUVEC microtissue-based angiogenesis. HUVEC microtissues (MTs), synthesized via hanging drop method, were subjected to angiogenesis in GTC-human fibrin (HF) hydrogels. Sprouting tip cells originated from the MTs within 24 h. Further, comprehensive in vitro study and in vivo validation revealed that, endothelial media with FBS and growth factors, 24 h old HUVEC MTs of 500 cells, seeded at 200 aggregates/cm3 in GTC-HF gel of 100 Pa elastic modulus, resulted in most optimal angiogenesis with intact lumen that was stable up to a week, without any supporting cells. Although early to predict, GTC-HF matrix may serve as a potential ECM for engineering complex and functional tissues of clinical relevance.

Ectopic vascularized bone formation by human mesenchymal stem cell microtissues in a biocomposite scaffold

Three-dimensional multicellular human bone marrow mesenchymal stem cells (hBM-MSCs) are showing a great promise in the repair of bone tissue due to its osteogenic differentiation potential, mimicking in vivo microenvironment and immunomodulatory property. In the present study, the potential of hBM-MSC microtissues (MTs) in combination with a biocomposite material to form vascularized bone-like tissue at an ectopic site in an immunocompromised mouse was evaluated. The scaffold was fabricated using gelatin, carboxymethyl cellulose, polyvinyl alcohol and nano-hydroxyapatite (GCnHP) by the freeze-drying method. The physico-chemico-biological characteristics were compared with control scaffold devoid of polyvinyl alcohol (GCnH). The scaffolds (GCnH and GCnHP) were highly porous and had interconnected pores. GCnHP showed higher mechanical strength, higher water adsorption and a lower rate of collagenase-mediated degradation in comparison to GCnH. The scaffolds also supported growth and proliferation of hBM-MSCs MTs and subsequent differentiation into osteoblast-like cells. The differentiated cells showed matrix mineralization and high expression of runX2, alkaline phosphatase, collagen type 1 and osteocalcin genes. A high expression of VEGF was also observed suggesting the potential of hBM-MSC MTs to induce angiogenesis. H&E and Masson's trichrome staining of the 4-weeks in vivo implanted scaffold revealed the presence of newly synthesized collagen and infiltration of host vasculature. IHC assessment showed expression of osteocalcin and osterix. These results demonstrate the efficacy of the combination of hBM-MSC MTs and biocomposite material as a promising approach for in vivo non-load bearing bone tissue repair for future clinical and various regenerative medicine applications.

Well-organized spheroids as a new platform to examine cell interaction and behaviour during organ development.

We present an experimental method allowing the production of three-dimensional organ-like structures, namely microtissues (MTs), in vitro without the need for exogenous extracellular matrix (ECM) or growth factors. Submandibular salivary glands (embryonic day ED14), kidneys (ED13) and lungs (ED13) were harvested from mouse embryos and dissociated into single cells by enzyme treatment. Single cells were seeded into special hanging drop culture plates (InSphero) and cultured for up to 14days to obtain MTs. This strategy permitted full control of the quantity of seeded cells. The development of the MTs into organs was followed histologically and immunohistochemically. Well-organized epithelial structures surrounded by a basal lamina were formed, as confirmed by transmission electron microscopy. Expression of E-cadherin, vimentin, fibronectin and α-SMA was compared in organs and corresponding MTs by real-time quantitative polymerase chain reaction. Branching morphogenesis was induced in MTs (as shown by histology and immunostaining for fibronectin and perlecan) and was conserved even after 14days of culture. MTs continued their development and their epithelial structures were comparable with those of the physiological organ at postnatal day 2 (PN2). Expression of aquaporins was investigated to obtain better support for the functional differentiation of epithelial cells. Histogenesis proceeded and led to the start of organogenesis. This experimental model might improve our knowledge of epithelial-mesenchymal histogenesis and can be employed to study development or cellular organization during the embryonic formation of organs.

Therapeutic Potential of Adipose-derived Stem Cell-based Microtissues in a Rat Model of Stress Urinary Incontinence

To examine the potential and mechanism of 3-dimensional cultures of adipose-derived stem cells (ADSCs) in the treatment of stress urinary incontinence (SUI) in a rat model simulating menopause combined with preceding childbirth injury. ADSCs were used to generate microtissues (MTs) with a hanging drop method. Forty-eight postpartum Sprague-Dawley rats were developed as SUI models after 4 hours of vagina dilation followed by bilateral ovariectomy. Ten rats that underwent sham ovariectomy without vagina dilation served as the control group. The SUI rats were divided into 3 groups and received urethral injection of phosphate-buffered saline, ADSCs, and MTs. Specimens were harvested for histology examination and ADSCs tracking at days 1, 3, 7, and 28 (n = 3) postinjection. At day 28, the remaining rats were examined for voiding function. Western blot, immunofluorescence, and immunohistochemistry staining were performed to examine histological changes and cytokine expression. The voiding function and histopathological structures were better recovered in the MT group than in the ADSC group. Compared with ADSCs, MTs express higher level of vascular endothelial growth factor and TNFα-stimulated gene/protein 6 in vitro, and represented a higher retention rate in vivo. Urethral injection of MTs better restored voiding function than ADSCs.

Therapeutic effects of adipose-derived stem cells-based microtissues on erectile dysfunction in streptozotocin-induced diabetic rats

This study aimed to explore the therapeutic effects of adipose-derived stem cells (ADSCs)-based microtissues (MTs) on erectile dysfunction (ED) in streptozotocin (STZ)-induced diabetic rats. Fifty-six 8-week-old Sprague-Dawley rats received intraperitoneal injection of STZ (60 mg kg−1), and 8 weeks later, the determined diabetic rats randomly received intracavernous (IC) injection of phosphate buffer solution (PBS), ADSCs, or MTs. Another eight normal rats equally got IC injection of PBS. MTs were generated with a hanging drop method, and the injected cells were tracked in ADSC- and MT-injected rats. Four weeks after the treatments, intracavernous pressure (ICP), histopathological changes in corpus cavernosum (CC), and functional proteins were measured. Rat cytokine antibody array was used to detect ADSCs or MTs lysate. The results showed that MTs expressed vascular endothelial growth factor (VEGF), nerve growth factor (NGF), and tumor necrosis factor-stimulated gene-6 (TSG-6). MTs injection had a higher retention than ADSCs injection and MTs treatment improved ICP, neuronal nitric oxide synthase (nNOS) expression, smooth muscle, and endothelial contents in diabetic rats, ameliorated local inflammation in CC better. Thus, our findings demonstrate that IC injection of MTs improves erectile function and histopathological changes in STZ-induced diabetic rats and appears to be more promising than traditional ADSCs. The underlying mechanisms involve increased cell retention accompanied with neuroprotection and anti-inflammatory behaviors of the paracrine factors.

AB240. Therapeutic effects of adipose-derived stem cells-based micro-tissues on erectile dysfunction in streptozotocin-induced diabetic rats

BackgroundThis study aimed to explore the therapeutic effects of adipose-derived stem cells (ADSCs)-based micro-tissues (MTs) on erectile dysfunction (ED) in streptozotocin (STZ) induced diabetic rats.MethodsFifty-six 8-week-old Sprague-Dawley rats received intraperitoneal injection of STZ (60 mg/kg) and eight weeks later, the determined diabetic rats randomly got intracavernous (IC) injection of phosphate buffer solution (PBS), ADSCs or MTs. Another eight normal rats equally received IC injection of PBS. MTs were generated with a hanging drop method and the injected cells were tracked in ADSCs and MTs injected rats. Four weeks after the treatments, intracavernous pressure (ICP), histopathological changes in corpus cavernosum (CC), and functional proteins were measured. Rat cytokine antibody array was used to detect ADSCs or MTs lysate.ResultsMTs expressed vascular endothelial growth factor (VEGF), nerve growth factor (NGF) and tumor necrosis factor-stimulated gene 6 (TSG-6). MTs injection had a higher retention than ADSCs injection and MTs treatment better improved ICP, neuronal nitric oxide synthase (nNOS) expression, smooth muscle and endothelial contents in diabetic rats, ameliorated local inflammation in CC.ConclusionsIC injection of MTs improves the erectile function and histopathological changes in streptozotocin-induced diabetic rats and appears to be more promising than traditional ADSCs. The underlying mechanisms involve increased cell retention accompanied with neuroprotection and anti-inflammatory behaviors of the paracrine factors.

AB185. Therapeutic potential of adipose-derived stem cells-based micro-tissues in a rat model of stress urinary incontinence

ObjectiveUsing adipose-derived stem cells (ADSCs) to halt stress urinary incontinence (SUI) progression and regenerate urethral sphincter (US) has emerged as a promising method to restore normal voiding function. However, traditional two-dimensional (2D) cell culture systems have limitations in mimicking in vivo conditions losing some important properties. This study aims to examine the potential and mechanism of three-dimensional (3D) cultures of ADSCs in the treatment of SUI in a rat model simulating childbirth injury.MethodsADSCs were used to generate micro-tissues (MTs) with a hanging drop method. A total of 48 postpartum Sprague-Dawley (SD) rats were developed SUI models by 4 h vagina dilation (VD) followed by bilateral ovariectomy (OV). Ten rats underwent sham OV without VD as control group. The SUI rats were divided into three groups and received urethral injection of PBS, ADSCs and MTs. specimens were harvested for histology and tracking CM-Dil-Labelled ADSCs at day 1, 3, 7 (n=3), and 28 (n=7) post-injection. At day 28, the remaining rats in each group were examined for voiding function. Western blot, immunofluorescence (IF) and immunohistochemistry (IHC) staining were performed to examine the histological change and cytokines.ResultsCompared with ADSCs, MTs expressed increased vascular endothelia growth factor (VEGF), TNFα stimulated gene/protein 6 (TSG-6) in vitro and represented a higher retention rate in vivo. The voiding function and histopathological recovery were significantly better in MTs group than ADSCs group.ConclusionsUrethral injection of MTs better restored the voiding function than ADSCs. The up-regulation of some bioactive factors and the improved cellular retention in the urethra may be responsible for the underlying mechanisms.

Development and Characterization of a Scaffold-Free 3D Spheroid Model of Induced Pluripotent Stem Cell-Derived Human Cardiomyocytes.

Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.

AB240. Therapeutic effects of adipose-derived stem cells-based micro-tissues on erectile dysfunction in streptozotocin-induced diabetic rats

ObjectiveThis study aimed to explore the therapeutic effects of adipose-derived stem cells (ADSCs)-based micro-tissues (MTs) on erectile dysfunction (ED) in streptozotocin (STZ) induced diabetic rats.MethodsFifty-eight 8-week-old Sprague-Dawley rats received intraperitoneal injection of STZ (60 mg/kg) and eight weeks later, the determined diabetic rats randomly got intracavernous (IC) injection of phosphate buffer solution (PBS), ADSCs and MTs. Another eight normal rats equally received IC injection of PBS. The injected cells were tracked in ADSCs and MTs groups. Four weeks after the treatment, intracavernous pressure (ICP), histopathological changes in corpus cavernosum (CC), apoptotic index and functional proteins were measured.ResultsMTs expressed vascular endothelial growth factor (VEGF), nerve growth factor (NGF), tissue inhibitor of metallopeptidase-1 (TIMP-1) and tumour necrosis factor-stimulated gene 6 (TSG-6). MTs had a better retention than ADSCs and MTs treatment improved ICP, neuronal nitric oxide synthase (nNOS) expression, smooth muscle and endothelial content in diabetic rats, ameliorated apoptosis and local inflammation in CC.ConclusionsThe results suggested that IC injection of MTs improves the erectile function and histopathological changes in STZ-induced diabetic rats and appears to be more promising than traditional ADSCs. The underlying mechanisms involve increased cell retention accompanied with neuroprotection and anti-inflammatory behaviors of the paracrine factors.

AB182. Therapeutic potential of adipose-derived stem cells-based micro-tissues in a postprostatectomy erectile dysfunction rat model

ObjectiveThis study aims to investigate the feasibility and mechanism of adipose-derived stem cells (ADSCs)-based micro-tissues (MTs) in the treatment of ED in a rat model of bilateral cavernous nerves (CNs) injury.MethodsADSCs labeled with 5-ethynyl-2-deoxyuridine (EdU) were used to generate MTs with hanging drop method. Ten Sprague-Dawley (SD) rats underwent sham surgery and intracavernous (IC) injection of phosphate buffer solution (PBS) (the sham group). Another 70 rats underwent bilateral CN crush and were then treated with PBS (n=10, the crush group), dissociated ADSCs (n=30, the ADSCs group), and MTs (n=30, the MTs group), respectively. At day 1, 3, 7, 14 (n=5), and 28 (n=10) postsurgery, specimens were harvested for histology. At day 28, 10 rats in each group were examined for erectile function before tissue harvest. Light microscopy of the dynamic aggregation of the MT, immunohistologic examination of the MTs, the retention and distribution of EdU + ADSCs in the corpus cavernosum (CC), and the penis histological analyses of collagen content, Western blot of functional proteins in MTs, IC pressure recording on CN electrostimulation.ResultsThree-day-old MTs became stable and expressed nerve growth factor, vascular endothelial growth factor, C-X-C chemokine receptor type 4, Wnt5a, and collagen IV. More EdU + ADSCs retained in the CC in the MTs group than that in the ADSCs group. IC injection of MTs resulted in significant restoration of the erectile function and histopathological changes compared with the ADSCs group.ConclusionsIC-injected MTs resulted in a better restoration of erectile function than traditional single-cell strategy. The underlying mechanisms of recovery appear to involve enhanced cellular retention in the penis and upregulation of some paracrine factors.

Therapeutic potential of adipose-derived stem cells-based micro-tissues in a rat model of postprostatectomy erectile dysfunction.

Stem cells (SCs) show significant benefits in the treatment of postprostatectomy erectile dysfunction (ED). However, the low retention rate of the traditional single-cell strategy at the injection sites limits its therapeutic potential. This study aims to investigate the feasibility and mechanism of adipose-derived stem cells (ADSCs)-based micro-tissues (MTs) in the treatment of ED in a rat model of bilateral cavernous nerves (CNs) injury. ADSCs labeled with 5-ethynyl-2-deoxyuridine (EdU) were used to generate MTs with hanging drop method. 10 Sprague-Dawley (SD) rats underwent sham surgery and intracavernous (IC) injection of phosphate buffer solution (PBS) (the sham group). Another 70 rats underwent bilateral CN crush and were then treated with PBS (n = 10, the crush group), dissociated ADSCs (n = 30, the ADSCs group), and MTs (n = 30, the MTs group), respectively. At day 1, 3, 7, 14 (n = 5), and 28 (n = 10) postsurgery, specimens were harvested for histology. At day 28, 10 rats in each group were examined for erectile function before tissue harvest. Light microscopy of the dynamic aggregation of the MT, immunohistologic examination of the MTs, the retention and distribution of EdU + ADSCs in the corpus cavernosum (CC), and the penis histological analyses of collagen content, Western blot of functional proteins in MTs, intracavernous pressure recording on CN electrostimulation. Three-day-old MTs became stable and expressed nerve growth factor, vascular endothelial growth factor, C-X-C chemokine receptor type 4, Wnt5a, and collagen IV. More EdU + ADSCs retained in the CC in the MTs group than that in the ADSCs group. IC injection of MTs resulted in significant restoration of the erectile function and histopathological changes compared with the ADSCs group. IC-injected MTs resulted in a better restoration of erectile function than traditional single-cell strategy. The underlying mechanisms of recovery appear to involve enhanced cellular retention in the penis and upregulation of some paracrine factors.

AB189. Therapeutic potential of adipose-derived stem cells based micro-tissues in a rat model of post-prostatectomy erectile dysfunction

IntroductionStem cells (SCs) show significant benefits in the treatment of post-prostatectomy erectile dysfunction (ED).However, the low retention rate of the tranditional single cell strategy at the injection sites limits its therapeutic potential.AimTo investigate the feasibility and mechanism of ADSCs based micro-tissues (MTs) in the treatment of ED in a rat model of bilateral cavernous nerves (CN) injury.MethodsADSCs labeled with 5-ethynyl-2-deoxyuridine (EdU) were used to generate MTs with hanging drop method. ten Sprague-Dawley (SD) rats underwent sham surgery and intracavernous (IC) injection of PBS (the sham group). Another 70 rats underwent bilateral CN crush and were then treated with PBS (n=10, the crush group), dissociated ADSCs (n=30, the ADSCs group), and MTs (n=30, the MTs group) respectively. At day 1, 3, 7, 14 (n=5) and 28 (n=10) post-surgery, specimens were harvested for histology. At day 28, 10 rats in each group were examined for erectile function before tissue harvest.Main outcome measuresLight microscopy of the dynamic aggregation of the micro-tissue, immunohistologic examination of the MTs, the retention and distribution of EdU + ADSCs in the corpus cavernosum (CC), and the penis histological analyses of collagen content, western blot of functional proteins in MTs, intracavernous pressure (ICP) recording on CN electrostimulation.ResultsThree-day old MTs became stable and expressed NGF, VEGF, CXCR4, Wnt5a, and ColIV. More EdU + ADSCs retained in the CC in the MTs group than that in the ADSCs group. IC injection of MTs resulted in significant restoration of the erectile function and histopathological changes compared to the ADSCs group.ConclusionsIC injected MTs resulted in a better restoration of erectile function than tranditional single cell strategy. The underlying mechanisms of recovery appear to involve enhanced cellular retention in the penis and up-regulation of some paracrine factors.

AB189. Therapeutic potential of adipose-derived stem cells based micro-tissues in a rat model of post-prostatectomy erectile dysfunction

IntroductionStem cells (SCs) show significant benefits in the treatment of post-prostatectomy erectile dysfunction (ED). However, the low retention rate of the traditional single cell strategy at the injection sites limits its therapeutic potential.AimTo investigate the feasibility and mechanism of ADSCs based micro-tissues (MTs) in the treatment of ED in a rat model of bilateral cavernous nerves (CN) injury.MethodsADSCs labeled with 5-ethynyl-2-deoxyuridine (EdU) were used to generate MTs with hanging drop method. 10 Sprague-Dawley (SD) rats underwent sham surgery and intracavernous (IC) injection of PBS (the sham group). Another 70 rats underwent bilateral CN crush and were then treated with PBS (n=10, the crush group), dissociated ADSCs (n=30, the ADSCs group), and MTs (n=30, the MTs group) respectively. At day 1, 3, 7, 14 (n=5) and 28 (n=10) postsurgery, specimens were harvested for histology. At day 28, 10 rats in each group were examined for erectile function before tissue harvest.Main outcome measuresLight microscopy of the dynamic aggregation of the micro-tissue, immunohistologic examination of the MTs, the retention and distribution of EdU+ ADSCs in the corpus cavernosum (CC), and the penishistological analyses of collagen content, western blot of functional proteins in MTs, intracavernous pressure (ICP) recording on CN electrostimulation.ResultsThree-day old MTs became stable and expressed NGF, VEGF, CXCR4, Wnt5a, and ColIV. More EdU+ ADSCs retained in the CC in the MTs group than that in the ADSCs group. IC injection of MTs resulted in significant restoration of the erectile function and histopathological changes compared to the ADSCs group.ConclusionIC injected MTs resulted in a better restoration of erectile function than tranditional single cell strategy. The underlying mechanisms of recovery appear to involve enhanced cellular retention in the penis and up-regulation of some paracrine factors.