Enhanced cellular engraftment of adipose-derived mesenchymal stem cell spheroids by using nanosheets as scaffolds.

The short survival time of transplanted adipose-derived mesenchymal stem cells (ASCs) is a problem for skin wound healing. Transplantation after the formation of cellular spheroids has been investigated as a promising method for prolonging cellular survival. However, there have been technical restrictions for transplantation of spheroids in clinical practice. Here, we show an effective method for transplantation of ASC spheroids onto skin wounds in order to efficiently cure refractory ulcers. To assist anchoring of spheroids onto skin wounds, we used a 120-nm-thick free-standing film (nanosheet) that has a highly adhesive property. Bioluminescence imaging showed that ASC spheroids carried by the nanosheet survived for 14 days, which is about two-times longer than that previously reported. Wounds treated with a nanosheet carrying ASC spheroids were 4-times smaller than untreated wounds on day 14. This method for transplantation of spheroids could be applied to cell therapy for various refractory skin wounds.

Transplantation of the Stromal Vascular Fraction (SVF) Mitigates Severe Radiation-Induced Skin Injury.

Severe radiation-induced skin injury is a complication of tumor radiotherapy and nuclear accidents. Cell therapy is a potential treatment for radiation-induced skin injury. The stromal vascular fraction (SVF) is a newer material in stem cell therapy that is made up of stem cells harvested from adipose tissue, which has been shown to promote the healing of refractory wounds of different causes. In this study, SVF was isolated from patients with radiation-induced skin injury. Adipose-derived stem cells (ADSCs) accounted for approximately 10% of the SVF by flow cytometry. Compared with the control group of rats, administration with SVF attenuated the skin injury induced by electron beam radiation. The effect of SVF on the human skin fibroblast microenvironment was determined by proteomic profiling of secreted proteins in SVF-co-cultured human skin fibroblast WS1 cells. Results revealed 293 upregulated and 1,481 downregulated proteins in the supernatant of SVF-co-cultured WS1 cells. WS1 co-culture with SVF induced secretion of multiple proteins including collagen and MMP-1. In the clinic, five patients with radiation-induced skin injury were recruited to receive SVF transfer-based therapy, either alone or combined with flap transplantation. Autogenous SVF was isolated and introduced into a multi-needle precision electronic injection device, which automatically and aseptically distributed the SVF to the exact layer of the wound in an accurate amount. After SVF transfer, wound healing clearly improved and pain was significantly relieved. The patients' skin showed satisfactory texture and shape with no further wound recurrence. Our findings suggest that transplantation of SVF could be an effective countermeasure against severe radiation-induced skin injury.

Human adipose-derived mesenchymal stem cell-based medical microrobot system for knee cartilage regeneration in vivo.

Targeted cell delivery by a magnetically actuated microrobot with a porous structure is a promising technique to enhance the low targeting efficiency of mesenchymal stem cell (MSC) in tissue regeneration. However, the relevant research performed to date is only in its proof-of-concept stage. To use the microrobot in a clinical stage, biocompatibility and biodegradation materials should be considered in the microrobot, and its efficacy needs to be verified using an in vivo model. In this study, we propose a human adipose-derived MSC-based medical microrobot system for knee cartilage regeneration and present an in vivo trial to verify the efficacy of the microrobot using the cartilage defect model. The microrobot system consists of a microrobot body capable of supporting MSCs, an electromagnetic actuation system for three-dimensional targeting of the microrobot, and a magnet for fixation of the microrobot to the damaged cartilage. Each component was designed and fabricated considering the accessibility of the patient and medical staff, as well as clinical safety. The efficacy of the microrobot system was then assessed in the cartilage defect model of rabbit knee with the aim to obtain clinical trial approval.

State of the Art Review of Cell Therapy in the Treatment of Lung Disease, and the Potential for Aerosol Delivery.

Respiratory and pulmonary diseases are among the leading causes of death globally. Despite tremendous advancements, there are no effective pharmacological therapies capable of curing diseases such as COPD (chronic obstructive pulmonary disease), ARDS (acute respiratory distress syndrome), and COVID-19. Novel and innovative therapies such as advanced therapy medicinal products (ATMPs) are still in early development. However, they have exhibited significant potential preclinically and clinically. There are several longitudinal studies published, primarily focusing on the use of cell therapies for respiratory diseases due to their anti-inflammatory and reparative properties, thereby hinting that they have the capability of reducing mortality and improving the quality of life for patients. The primary objective of this paper is to set out a state of the art review on the use of aerosolized MSCs and their potential to treat these incurable diseases. This review will examine selected respiratory and pulmonary diseases, present an overview of the therapeutic potential of cell therapy and finally provide insight into potential routes of administration, with a focus on aerosol-mediated ATMP delivery.

Protective effect of brain-derived neurotrophic factor and neurotrophin-3 overexpression by adipose-derived stem cells combined with silk fibroin/chitosan scaffold in spinal cord injury.

Objectives: Spinal cord injury (SCI) is a most debilitating traumatic injury, and cytotherapy is a promising alternative treatment strategy. Here we investigated the effect and mechanism of adipose-derived stem/stromal cells (ASCs) with overexpressing brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT3) (BDNF-NT3) in combination with silk fibroin/chitosan scaffold (SFCS) in SCI.Methods: Female Sprague-Dawley rats were used as an SCI model. SFCS,SFCS and ASCs, or ASCs overexpressing NT3, BDNF, and BDNF-NT3 were implanted into SCI rats. Basso, Beattie, and Bresnahan score, pathological changes, and spinal cord tissue and nerve fiber morphology were observed and assayed. GAP-43, GFAP, and caspase-3 expression was determined using immunohistochemistry and western blotting.Results: Smoother spinal cords, less scar tissue, and lower inflammatory activity were found in the SFCS, SFCS and ASCs, ASCs with NT3, BDNF, and BDNF-NT3 overexpression treatment than in the untreated SCI rat groups. Increasing formation of nerve fibers was observed in the above groups in order. GAP-43 expression significantly increased, while GFAP and caspase-3 expression significantly decreased. These results indicated obvious alleviation in pathological changes and BDNF-NT3 overexpression in ASCs combined with SFCS treatment in SCI rats.Conclusion: Thus, BDNF-NT3 overexpression from ASCs with SFCS had synergistic neuroprotective effects on SCI and may be a treatment option for SCI.

Treatment of rats with spinal cord injury using human bone marrow-derived stromal cells prepared by negative selection.

BACKGROUND: Spinal cord injury (SCI) is a highly debilitating pathology without curative treatment. One of the most promising disease modifying strategies consists in the implantation of stem cells to reduce inflammation and promote neural regeneration. In the present study we tested a new human bone marrow-derived stromal cell preparation (bmSC) as a therapy of SCI. METHODS: Spinal cord contusion injury was induced in adult male rats at thoracic level T9/T10 using the Infinite Horizon impactor. One hour after lesion the animals were treated with a sub-occipital injection of human bmSC into the cisterna magna. No immune suppression was used. One dose of bmSC consisted, on average, of 2.3 million non-manipulated cells in 100 µL suspension, which was processed out of fresh human bone marrow from the iliac crest of healthy volunteers. Treatment efficacy was compared with intraperitoneal injections of methylprednisolone (MP) and saline. The recovery of motor functions was assessed during a surveillance period of nine weeks. Adverse events as well as general health, weight and urodynamic functions were monitored daily. After this time, the animals were perfused, and the spinal cord tissue was investigated histologically. RESULTS: Rats treated with bmSC did not reject the human implants and showed no sign of sickness behavior or neuropathic pain. Compared to MP treatment, animals displayed better recovery of their SCI-induced motor deficits. There were no significant differences in the recovery of bladder control between groups. Histological analysis at ten weeks after SCI revealed no differences in tissue sparing and astrogliosis, however, bmSC treatment was accompanied with reduced axonal degeneration in the dorsal ascending fiber tracts, lower Iba1-immunoreactivity (IR) close to the lesion site and reduced apoptosis in the ventral grey matter. Neuroinflammation, as evidenced by CD68-IR, was significantly reduced in the MP-treated group. CONCLUSIONS: Human bmSC that were prepared by negative selection without expansion in culture have neuroprotective properties after SCI. Given the effect size on motor function, implantation in the acute phase was not sufficient to induce spinal cord repair. Due to their immune modulatory properties, allogeneic implants of bmSC can be used in combinatorial therapies of SCI.

Human intracerebroventricular (ICV) injection of autologous, non-engineered, adipose-derived stromal vascular fraction (ADSVF) for neurodegenerative disorders: results of a 3-year phase 1 study of 113 injections in 31 patients.

We have chosen to test the safety of human intracerebroventricular (ICV) brain injections of autologous non-genetically-modified adipose-derived stromal vascular fraction (ADSVF). In this IRB-approved trial, 24 patients received ICV ADSVF via an implanted reservoir between 5/22/14 and 5/22/17. Seven others were injected via their ventriculo-peritoneal shunts. Ten patients had Alzheimer's disease (AD), 6 had amyotrophic lateral sclerosis (ALS), 6 had progressive multiple sclerosis (MS-P), 6 had Parkinson's "Plus" (PD+), 1 had spinal cord injury, 1 had traumatic brain injury, and 1 had stroke. Median age was 74 (range 41-83). Injections were planned every 2-3 months. Thirty-one patients had 113 injections. Patients received SVF injection volumes of 3.5-20 cc (median:4 cc) containing 4.05 × 105 to 6.2 × 107 cells/cc, which contained an average of 8% hematopoietic and 7.5% adipose stem cells. Follow-up ranged from 0 to 36 months (median: 9.2 months). MRIs post injection(s) were unchanged, except for one AD patient whose hippocampal volume increased from < 5th percentile to 48th percentile (NeuroQuant® volumetric MRI). Of the 10 AD patients, 8 were stable or improved in tests of cognition. Two showed improvement in P-tau and ß-amyloid levels. Of the 6 MS-P patients all are stable or improved. Four of 6 ALS patients died of disease progression. Twelve of 111 injections (11%) led to 1-4 days of transient meningismus, and mild temperature elevation, which resolved with acetaminophen and/or dexamethasone. Two (1.8% of injections) required hospitalization for these symptoms. One patient (0.9% of injections) had his reservoir removed and later replaced for presumed infection. In this Phase 1 safety trial, ADSVF was safely injected into the human brain ventricular system in patients with no other treatment options. Secondary endpoints of clinical improvement or stability were particularly promising in the AD and MS-P groups. These results will be submitted for a Phase 2 FDA-approved trial.

Infusing Mesenchymal Stromal Cells into Porcine Kidneys during Normothermic Machine Perfusion: Intact MSCs Can Be Traced and Localised to Glomeruli.

Normothermic machine perfusion (NMP) of kidneys offers the opportunity to perform active interventions, such as the addition of mesenchymal stromal cells (MSCs), to an isolated organ prior to transplantation. The purpose of this study was to determine whether administering MSCs to kidneys during NMP is feasible, what the effect of NMP is on MSCs and whether intact MSCs are retained in the kidney and to which structures they home. Viable porcine kidneys were obtained from a slaughterhouse. Kidneys were machine perfused during 7 h at 37 °C. After 1 h of perfusion either 0, 105, 106 or 107 human adipose tissue derived MSCs were added. Additional ex vivo perfusions were conducted with fluorescent pre-labelled bone-marrow derived MSCs to assess localisation and survival of MSCs during NMP. After NMP, intact MSCs were detected by immunohistochemistry in the lumen of glomerular capillaries, but only in the 107 MSC group. The experiments with fluorescent pre-labelled MSCs showed that only a minority of glomeruli were positive for infused MSCs and most of these glomeruli contained multiple MSCs. Flow cytometry showed that the number of infused MSCs in the perfusion circuit steeply declined during NMP to approximately 10%. In conclusion, the number of circulating MSCs in the perfusate decreases rapidly in time and after NMP only a small portion of the MSCs are intact and these appear to be clustered in a minority of glomeruli.

Mesenchymal Stem Cell Capping on ECM-Anchored Caspase Inhibitor-Loaded PLGA Microspheres for Intraperitoneal Injection in DSS-Induced Murine Colitis.

Mesenchymal stem cells (MSCs) are considered as a promising alternative for the treatment of various inflammatory disorders. However, poor viability and engraftment of MSCs after transplantation are major hurdles in mesenchymal stem cell therapy. Extracellular matrix (ECM)-coated scaffolds provide better cell attachment and mechanical support for MSCs after transplantation. A single-step method for ECM functionalization on poly(lactic-co-glycolic acid) (PLGA) microspheres using a novel compound, dopamine-conjugated poly(ethylene-alt-maleic acid), as a stabilizer during the preparation of microspheres is reported. The dopamine molecules on the surface of microspheres provide active sites for the conjugation of ECM in an aqueous solution. The results reveal that the viability of MSCs improves when they are coated over the ECM-functionalized PLGA microspheres (eMs). In addition, the incorporation of a broad-spectrum caspase inhibitor (IDN6556) into the eMs synergistically increases the viability of MSCs under in vitro conditions. Intraperitoneal injection of the MSC-microsphere hybrid alleviates experimental colitis in a murine model via inhibiting Th1 and Th17 differentiation of CD4+ T cells in colon-draining mesenteric lymph nodes. Therefore, drug-loaded ECM-coated surfaces may be considered as attractive tools for improving viability, proliferation, and functionality of MSCs following transplantation.

A biomaterials approach to influence stem cell fate in injectable cell-based therapies.

BACKGROUND: Numerous stem cell therapies use injection-based administration to deliver high-density cell preparations. However, cell retention rates as low as 1% have been observed within days of transplantation. This study investigated the effects of varying administration and formulation parameters of injection-based administration on cell dose recovery and differentiation fate choice of human mesenchymal stem cells. METHODS: The impact of ejection rate via clinically relevant Hamilton micro-syringes and biomaterial-assisted delivery was investigated. Cell viability, the percentage of cell dose delivered as viable cells, proliferation capacity as well as differentiation behaviour in bipotential media were assessed. Characterisation of the biomaterial-based cell carriers was also carried out. RESULTS: A significant improvement of in-vitro dose recovery in cells co-ejected with natural biomaterials was observed, with ejections within 2% (w/v) gelatin resulting in 87.5 ± 14% of the cell dose being delivered as viable cells, compared to 32.2 ± 19% of the dose ejected in the commonly used saline vehicle at 10 µl/min. Improvement in cell recovery was not associated with the rheological properties of biomaterials utilised, as suggested by previous studies. The extent of osteogenic differentiation was shown to be substantially altered by choice of ejection rate and cell carrier, despite limited contact time with cells during ejection. Collagen type I and bone-derived extracellular matrix cell carriers yielded significant increases in mineralised matrix deposited at day 21 relative to PBS. CONCLUSIONS: An enhanced understanding of how administration protocols and biomaterials influence cell recovery, differentiation capacity and choice of fate will facilitate the development of improved administration and formulation approaches to achieve higher efficacy in stem cell transplantation.

Human adipose-derived stem cells and simvastatin-functionalized biomimetic calcium phosphate to construct a novel tissue-engineered bone.

To repair bone defects, we evaluate the in-vitro and in-vivo osteogenic activities of a novel tissue-engineered bone (TEB) by elaborately combining biomimetic calcium phosphate (BioCaP) granules with internally-incorporated simvastatin (SIM) and human adipose-derived stem cells (hASCs). First, we constructed BioCaP with SIM internally incorporated (SIM-BioCaP). Then we characterized the morphology and chemical composition of SIM-BioCaP. The release kinetics of SIM was monitored in vitro spectroscopically. Thereafter, we explored the in-vitro cellular responses of hASCs to SIM-BioCaP by performing scanning electron microscopy observation, proliferation assay, alkaline phosphatase (ALP) activity assay, alizarin red staining and real-time PCR. Finally, we investigated the in-vivo osteogenic activities of the novel TEB in a subcutaneous bone induction model in nude mice. We found that SIM was successfully incorporated internally in BioCaP and showed a slow release manner without significantly affecting the attachment and proliferation of hASCs. The released SIM from BioCaP could significantly enhance the proliferation, ALP activities, mineralized nodules formation and osteogenic genes of hASCs. The in-vivo tests showed this TEB could induce new bone formation while the other groups could not. Taken together, the present data show that this novel TEB represented a very promising construct to treat critical-volume bone defects.

A novel cytotherapy device for rapid screening, enriching and combining mesenchymal stem cells into a biomaterial for promoting bone regeneration.

Bone defects are a common challenge in clinic, usually warranting bone grafts. However, current strategies to obtain effective graft materials have many drawbacks. Mesenchymal stem cell (MSC)-based therapy is a promising alternative. We designed an innovative appliance named the stem cell screen-enrich-combine(-biomaterials) circulating system (SECCS). In this study, 42 patients who required bone graft underwent SECCS-based treatment. Their bone marrow samples and beta-tricalcium phosphate (ß-TCP) granules were processed in the SECCS for 10-15 minutes, to produce MSC/ß-TCP composites. These composites were grafted back into bone defect sites. The results showed 85.53% ± 7.95% autologous MSCs were successfully screened, enriched, and seeded on the ß-TCP scaffolds synchronously. The cell viability remained unchanged after SECCS processing. Clinically, all patients obtained satisfactory bone healing. Thus, without in vitro culture, the SECCS can produce bioactive MSC/ß-TCP composites for bone regeneration during surgery. The SECCS represents a convenient, rapid, low-cost, and safe method for bone regeneration.

Mesenchymal stem cell transplantation carried in SVVYGLR modified self-assembling peptide promoted cardiac repair and angiogenesis after myocardial infarction.

The efficiency of stem cell therapy for myocardial infarction (MI) was very low due to the hostile microenvironment and poor blood perfusion. In this study, we designed a new self-assembling peptide through adding angiogenic polypeptide SVVYGLR to the carboxyl terminal of RADA16, and evaluated the therapeutic potential of mesenchymal stem cell (MSC) transplantation carried in this designer self-assembling peptide (DSP) on MI. After the model of cell ischemia and hypoxia was established in vitro, cytoprotective effect of DSP on MSC was detected by AO/EB staining. MI was induced by ligating of the left anterior descending artery in female SD rats. MSC from male rats was labled by GFP with adenovirus transfection. MSC with DSP (MSC-DSP) or without DSP (MSC) were transplanted at the border of the infarcted area. The number of survival cell was more and necrotic cell was less in DSP group than that in control group after ischemia and hypoxia treatment in vitro. At 4 weeks after cell transplantation, compared with the MSC group, improvement of cardiac function was better, infarct size was reduced, collagen content and the number of apoptotic cells was decreased, and there were more GFP or SRY positive cells in MSC-DSP group. Moreover, the number of CD31 or α-smooth muscle actin positive blood vessels in MSC-DSP group was significantly higher than that in MSC group. DSP not only provided a microenvironment for the survival of MSC, but also promoted the angiogenesis after transplantation. This study provided novel strategy and experimental evidence for the clinical application of biomaterials in stem cell transplantation for treatment of ischemic heart disease such as MI.

A chitosan/dextran-based hydrogel as a delivery vehicle of human bone-marrow derived mesenchymal stem cells.

A chitosan/dextran-based (CD) injectable, surgical hydrogel has been developed and shown to be an effective post-operative aid in prevention of scar tissue formation in vivo. The CD hydrogel's effectiveness in a surgical setting prompted an investigation into its capacity as a potential delivery vehicle for bone marrow derived mesenchymal stem cells (BM-MSCs) for regenerative wound healing applications. By housing BM-MSCs within a biocompatible, injectable, hydrogel matrix, viability and protection in cultivation, as well as direct delivery to the damaged site in the host tissue may be achieved. In vitro BM-MSC cell viability in the presence of CD hydrogel was determined by LIVE/DEAD® fluoresence staining. Flow cytometry studies revealed expression of a conventional BM-MSC surface marker profile. A colony forming cell assay showed a slight statistically significant decrease in the number of colonies grown in CD hydrogel as compared to control cells. In addition, BM-MSCs in the CD hydrogel were able to successfully differentiate into adipocytes and osteocytes. In summary, the CD hydrogel supports MSC growth and differentiation; and therefore, may be used as a potential stem cell delivery vehicle for regenerative medicine and tissue engineering applications.

Development of an optical system for the non-invasive tracking of stem cell growth on microcarriers.

The emergence of medicinal indications for stem cell therapies has seen a need to develop the manufacturing capacity for adherent cells such as mesenchymal stem cells (MSCs). One such development is in the use of microcarriers, which facilitate enhanced cell densities for adherent stem cell cultures when compared with 2D culture platforms. Given the variety of stem cell expansion systems commercially available, novel methods of non-invasive and automated monitoring of cell number, confluence, and aggregation, within disparate environments, will become imperative to process control, ensuring reliable and consistent performance. The in situ epi-illumination of mouse embryonic fibroblasts and human mesenchymal stem cells attached to Cytodex 1 and 3 microcarriers was achieved using a bespoke microscope. Robust image processing techniques were developed to provide quantitative measurements of confluence, aggregate recognition, and cell number, without the need for fluorescent labeling or cell detachment. Large datasets of cells counted on individual microcarriers were statistically analyzed and compared with NucleoCounter measurements, with an average difference of less than 7% observed from days 0 to 6 of a 12-day culture noted, prior to the onset of aggregation. The developed image acquisition system and post-processing methodologies were successfully applied to dynamically moving colonized microcarriers. The proposed system offers a novel method of cell identification at the individual level, to consistently and accurately assess viable cell number, confluence, and cell distribution, while also minimizing the variability inherent in the current invasive means by which cells adhered to microcarriers are analyzed. Biotechnol. Bioeng. 2017;114: 2032-2042. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.

3D-Printed Poly(ε-caprolactone) Scaffold Augmented With Mesenchymal Stem Cells for Total Meniscal Substitution: A 12- and 24-Week Animal Study in a Rabbit Model.

BACKGROUND: Total meniscectomy leads to knee osteoarthritis in the long term. The poly(ε-caprolactone) (PCL) scaffold is a promising material for meniscal tissue regeneration, but cell-free scaffolds result in relatively poor tissue regeneration and lead to joint degeneration. HYPOTHESIS: A novel, 3-dimensional (3D)-printed PCL scaffold augmented with mesenchymal stem cells (MSCs) would offer benefits in meniscal regeneration and cartilage protection. STUDY DESIGN: Controlled laboratory study. METHODS: PCL meniscal scaffolds were 3D printed and seeded with bone marrow-derived MSCs. Seventy-two New Zealand White rabbits were included and were divided into 4 groups: cell-seeded scaffold, cell-free scaffold, sham operation, and total meniscectomy alone. The regeneration of the implanted tissue and the degeneration of articular cartilage were assessed by gross and microscopic (histological and scanning electron microscope) analysis at 12 and 24 weeks postoperatively. The mechanical properties of implants were also evaluated (tensile and compressive testing). RESULTS: Compared with the cell-free group, the cell-seeded scaffold showed notably better gross appearance, with a shiny white color and a smooth surface. Fibrochondrocytes with extracellular collagen type I, II, and III and proteoglycans were found in both seeded and cell-free scaffold implants at 12 and 24 weeks, while the results were significantly better for the cell-seeded group at week 24. Furthermore, the cell-seeded group presented notably lower cartilage degeneration in both femur and tibia compared with the cell-free or meniscectomy group. Both the tensile and compressive properties of the implants in the cell-seeded group were significantly increased compared with those of the cell-free group. CONCLUSION: Seeding MSCs in the PCL scaffold increased its fibrocartilaginous tissue regeneration and mechanical strength, providing a functional replacement to protect articular cartilage from damage after total meniscectomy. CLINICAL RELEVANCE: The study suggests the potential of the novel 3D PCL scaffold augmented with MSCs as an alternative meniscal substitution, although this approach requires further improvement before being used in clinical practice.

Automated freeze-thaw cycles for decellularization of tendon tissue - a pilot study.

BACKGROUND: Decellularization of tendon tissue plays a pivotal role in current tissue engineering approaches for in vitro research as well as for translation of graft-based tendon restoration into clinics. Automation of essential decellularization steps like freeze-thawing is crucial for the development of more standardized decellularization protocols and commercial graft production under good manufacturing practice (GMP) conditions in the future. METHODS: In this study, a liquid nitrogen-based controlled rate freezer was utilized for automation of repeated freeze-thawing for decellularization of equine superficial digital flexor tendons. Additional tendon specimens underwent manually performed freeze-thaw cycles based on an established procedure. Tendon decellularization was completed by using non-ionic detergent treatment (Triton X-100). Effectiveness of decellularization was assessed by residual nuclei count and calculation of DNA content. Cytocompatibility was evaluated by culturing allogeneic adipose tissue-derived mesenchymal stromal cells on the tendon scaffolds. RESULTS: There were no significant differences in decellularization effectiveness between samples decellularized by the automated freeze-thaw procedure and samples that underwent manual freeze-thaw cycles. Further, we inferred no significant differences in the effectiveness of decellularization between two different cooling and heating rates applied in the automated freeze-thaw process. Both the automated protocols and the manually performed protocol resulted in roughly 2% residual nuclei and 13% residual DNA content. Successful cell culture was achieved with samples decellularized by automated freeze-thawing as well as with tendon samples decellularized by manually performed freeze-thaw cycles. CONCLUSIONS: Automated freeze-thaw cycles performed by using a liquid nitrogen-based controlled rate freezer were as effective as previously described manual freeze-thaw procedures for decellularization of equine superficial digital flexor tendons. The automation of this key procedure in decellularization of large tendon samples is an important step towards the processing of large sample quantities under standardized conditions. Furthermore, with a view to the production of commercially available tendon graft-based materials for application in human and veterinary medicine, the automation of key procedural steps is highly required to develop manufacturing processes under GMP conditions.

Sulfated hyaluronic acid hydrogels with retarded degradation and enhanced growth factor retention promote hMSC chondrogenesis and articular cartilage integrity with reduced hypertrophy.

Recently, hyaluronic acid (HA) hydrogels have been extensively researched for delivering cells and drugs to repair damaged tissues, particularly articular cartilage. However, the in vivo degradation of HA is fast, thus limiting the clinical translation of HA hydrogels. Furthermore, HA cannot bind proteins with high affinity because of the lack of negatively charged sulfate groups. In this study, we conjugated tunable amount of sulfate groups to HA. The sulfated HA exhibits significantly slower degradation by hyaluronidase compared to the wild type HA. We hypothesize that the sulfation reduces the available HA octasaccharide substrate needed for the effective catalytic action of hyaluronidase. Moreover, the sulfated HA hydrogels significantly improve the protein sequestration, thereby effectively extending the availability of the proteinaceous drugs in the hydrogels. In the following in vitro study, we demonstrate that the HA hydrogel sulfation exerts no negative effect on the viability of encapsulated human mesenchymal stem cells (hMSCs). Furthermore, the sulfated HA hydrogels promote the chondrogenesis and suppresses the hypertrophy of encapsulated hMSCs both in vitro and in vivo. Moreover, intra-articular injections of the sulfated HA hydrogels avert the cartilage abrasion and hypertrophy in the animal osteoarthritic joints. Collectively, our findings demonstrate that the sulfated HA is a promising biomaterial for the delivery of therapeutic agents to aid the regeneration of injured or diseased tissues and organs. STATEMENT OF SIGNIFICANCE: In this paper, we conjugated sulfate groups to hyaluronic acid (HA) and demonstrated the slow degradation and growth factor delivery of sulfated HA. Furthermore, the in vitro and in vivo culture of hMSCs laden HA hydrogels proved that the sulfation of HA hydrogels not only promotes the chondrogenesis of hMSCs but also suppresses hypertrophic differentiation of the chondrogenically induced hMSCs. The animal OA model study showed that the injected sulfated HA hydrogels significantly reduced the cartilage abrasion and hypertrophy in the animal OA joints. We believe that this study will provide important insights into the design and optimization of the HA-based hydrogels as the scaffold materials for cartilage regeneration and OA treatment in clinical setting.

Gelatin-based 3D conduits for transdifferentiation of mesenchymal stem cells into Schwann cell-like phenotypes.

In this study, gelatin-based 3D conduits with three different microstructures (nanofibrous, macroporous and ladder-like) were fabricated for the first time via combined molding and thermally induced phase separation (TIPS) technique for peripheral nerve regeneration. The effects of conduit microstructure and mechanical properties on the transdifferentiation of bone marrow-derived mesenchymal stem cells (MSCs) into Schwann cell (SC) like phenotypes were examined to help facilitate neuroregeneration and understand material-cell interfaces. Results indicated that 3D macroporous and ladder-like structures enhanced MSC attachment, proliferation and spreading, creating interconnected cellular networks with large numbers of viable cells compared to nanofibrous and 2D-tissue culture plate counterparts. 3D-ladder-like conduit structure with complex modulus of ∼0.4×106Pa and pore size of ∼150µm provided the most favorable microenvironment for MSC transdifferentiation leading to ∼85% immunolabeling of all SC markers. On the other hand, the macroporous conduits with complex modulus of ∼4×106Pa and pore size of ∼100µm showed slightly lower (∼65% for p75, ∼75% for S100 and ∼85% for S100ß markers) immunolabeling. Transdifferentiated MSCs within 3D-ladder-like conduits secreted significant amounts (∼2.5pg/mL NGF and ∼0.7pg/mL GDNF per cell) of neurotrophic factors, while MSCs in macroporous conduits released slightly lower (∼1.5pg/mL NGF and 0.7pg/mL GDNF per cell) levels. PC12 cells displayed enhanced neurite outgrowth in media conditioned by conduits with transdifferentiated MSCs. Overall, conduits with macroporous and ladder-like 3D structures are promising platforms in transdifferentiation of MSCs for neuroregeneration and should be further tested in vivo. STATEMENT OF SIGNIFICANCE: This manuscript focuses on the effect of microstructure and mechanical properties of gelatin-based 3D conduits on the transdifferentiation of mesenchymal stem cells to Schwann cell-like phenotypes. This work builds on our recently accepted manuscript in Acta Biomaterialia focused on multifunctional 2D films, and focuses on 3D microstructured conduits designed to overcome limitations of current strategies to facilitate peripheral nerve regeneration. The comparison between conduits fabricated with nanofibrous, macroporous and ladder-like microstructures showed that the ladder-like conduits showed the most favorable environment for MSC transdifferentiation to Schwann-cell like phenotypes, as seen by both immunolabeling as well as secretion of neurotrophic factors. This work demonstrates the importance of controlling the 3D microstructure to facilitate tissue engineering strategies involving stem cells that can serve as promising approaches for peripheral nerve regeneration.