Prevascularized hydrogels with mature vascular networks promote the regeneration of critical-size calvarial bone defects in vivo.

Adequate vascularization of scaffolds is a prerequisite for successful repair and regeneration of lost and damaged tissues. It has been suggested that the maturity of engineered vascular capillaries, which is largely determined by the presence of functional perivascular mural cells (or pericytes), plays a vital role in maintaining vessel integrity during tissue repair and regeneration. Here, we investigated the role of pericyte-supported-engineered capillaries in regenerating bone in a critical-size rat calvarial defect model. Prior to implantation, human umbilical vein endothelial cells and human bone marrow stromal cells (hBMSCs) were cocultured in a collagen hydrogel to induce endothelial cell morphogenesis into microcapillaries and hBMSC differentiation into pericytes. Upon implantation into the calvarial bone defects (8 mm), the prevascularized hydrogels showed better bone formation than either untreated controls or defects treated with autologous bone grafts (positive control). Bone formation parameters such as bone volume, coverage area, and vascularity were significantly better in the prevascularized hydrogel group than in the autologous bone group. Our results demonstrate that tissue constructs engineered with pericyte-supported vascular capillaries may approximate the regenerative capacity of autologous bone, despite the absence of osteoinductive or vasculogenic growth factors.

Effectiveness of a biodegradable 3D polylactic acid/poly(ɛ-caprolactone)/hydroxyapatite scaffold loaded by differentiated osteogenic cells in a critical-sized radius bone defect in rat.

The effects of a scaffold made of polylactic acid, poly (ɛ-caprolactone) and hydroxyapatite by indirect 3D printing method with and without differentiated bone cells was tested on the regeneration of a critical radial bone defect in rat. The scaffold characterization and mechanical performance were determined by the rheology, scanning electron microscopy, energy-dispersive X-ray spectroscopy, X-ray diffraction, and Fourier transform infrared spectrometry. The defects were created in forty Wistar rats which were randomly divided into the untreated, autograft, scaffold cell-free, and differentiated bone cell-seeded scaffold groups (n = 10 in each group). The expression level of angiogenic and osteogenic markers, analyzed by quantitative real time-polymerase chain reaction (in vitro), significantly improved (p < 0.05) in the scaffold group compared to the untreated one. Radiology and computed tomography scan demonstrated a significant improvement in the cell-seeded scaffold group compared to the untreated one (p < 0.001). Biomechanical, histopathological, histomorphometric, and immunohistochemical investigations showed significantly better regeneration scores in the cell-seeded scaffold and autograft groups compared to the untreated group (p < 0.05). The cell-seeded scaffold and autograft groups did show comparable results on the 80th day post-treatment (p > 0.05), however, most results in the scaffold group were significantly higher than the untreated group (p < 0.05). Differentiated bone cells can enhance bone regeneration potential of the scaffold.

Validation of an implantable bioink using mechanical extraction of human skin cells: First steps to a 3D bioprinting treatment of deep second degree burn.

Clinical grade cultured epithelial autograft (CEA) are routinely used to treat burns covering more than 60% of the total body surface area. However, although the epidermis may be efficiently repaired by CEA, the dermal layer, which is not spared in deep burns, requires additional treatment strategies. Our aim is to develop an innovative method of skin regeneration based on in situ 3D bioprinting of freshly isolated autologous skin cells. We describe herein bioink formulation and cell preparation steps together with experimental data validating a straightforward enzyme-free protocol of skin cell extraction. This procedure complies with both the specific needs of 3D bioprinting process and the stringent rules of good manufacturing practices. This mechanical extraction protocol, starting from human skin biopsies, allows harvesting a sufficient amount of both viable and growing keratinocytes and fibroblasts. We demonstrated that a dermis may be reconstituted in vitro starting from a medical grade bioink and mechanically extracted skin cells. In these experiments, proliferation of the extracted cells can be observed over the first 21 days period after 3D bioprinting and the analysis of type I collagen exhibited a de novo production of extracellular matrix proteins. Finally, in vivo experiments in a murine model of severe burn provided evidences that a topical application of our medical grade bioink was feasible and well-tolerated. Overall, these results represent a valuable groundwork for the design of future 3D bioprinting tissue engineering strategies aimed at treating, in a single intraoperative step, patients suffering from extended severe burns.

Proteomic profiling of striatal tissue of a rat model of Parkinson's disease after implantation of collagen-encapsulated human umbilical cord mesenchymal stem cells.

Parkinson's disease (PD) is the most common neurodegenerative disorder of movement worldwide. To date, only symptomatic treatments are available. Implantation of collagen-encapsulated human umbilical cord mesenchymal stem cells (hUC-MSCs) is being developed as a novel therapeutic approach to potentially modify PD progression. However, implanted collagen scaffolds may induce a host tissue response. To gain insight into such response, hUC-MSCs were encapsulated into collagen hydrogels and implanted into the striatum of hemi-Parkinsonian male Sprague-Dawley rats. One or 14 days after implantation, the area of interest was dissected using a cryostat. Total protein extracts were subjected to tryptic digestion and subsequent LC-MS/MS analyses for protein expression profiling. Univariate and multivariate analyses were performed to identify differentially expressed protein profiles with subsequent gene ontology and pathway analysis for biological interpretation of the data; 2,219 proteins were identified by MaxQuant at 1% false discovery rate. A high correlation of label-free quantification (LFQ) protein values between biological replicates (r = .95) was observed. No significant differences were observed between brains treated with encapsulated hUC-MSCs compared to appropriate controls. Proteomic data were highly robust and reproducible, indicating the suitability of this approach to map differential protein expression caused by the implants. The lack of differences between conditions suggests that the effects of implantation may be minimal. Alternatively, effects may only have been focal and/or could have been masked by nonrelevant high-abundant proteins. For follow-up assessment of local changes, a more accurate dissection technique, such as laser micro dissection, and analysis method are recommended.

Collagen-tussah silk fibroin hybrid scaffolds loaded with bone mesenchymal stem cells promote skin wound repair in rats.

This study demonstrates the efficacy of collagen/tussah silk fibroin (Col/TSF) hybrid scaffolds loaded with bone mesenchymal stem cells (BMSCs) in skin repair. Collagen (Col) and tussah silk fibroin (TSF) were extracted from bovine tendons and tussah cocoons, respectively. Col/TSF scaffolds were obtained using a freeze-drying method and were characterised using fourier transform infrared spectroscopy, scanning electron microscopy, porosity, water retention, thermal stability, and biocompatibility. The results revealed that addition of TSF to scaffolds could enhance their moisturising ability and cell infiltration. The antibacterial properties of Col/TSF scaffolds loaded with antibiotics were also excellent. BMSCs cultured in contact with developed Col/TSF scaffolds showed increased cell adhesion, viability, and differentiation. An in vivo study on rats showed that the Col/TSF scaffold seeded with BMSCs was more conducive to wound healing compared to the Col/TSF scaffold alone. The present study suggests that Col/TSF scaffold seeded with BMSCs could be a promising candidate for skin tissue engineering, due to its excellent skin affinity, good air and water permeability, and improved wound healing potential.

Accelerating bone defects healing in calvarial defect model using 3D cultured bone marrow-derived mesenchymal stem cells on demineralized bone particle scaffold.

Bone defects are usually difficult to be regenerated due to pathological states or the size of the injury. Researchers are focusing on tissue engineering approaches in order to drive the regenerative events, using stem cells to regenerate bone. The purpose of this study is to evaluate the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) on biologically derived Gallus gallus domesticus-derived demineralized bone particle (GDD) sponge. The sponges were prepared by freeze-drying method using 1, 2, and 3 wt% GDD and cross-linked with glutaraldehyde. The GDD sponge was characterized using scanning electron microscopy, compressive strength, porosity, and Fourier transform infrared. The potential bioactivity of the sponge was evaluated by osteogenic differentiation of BMSCs using 3(4, dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and quantifying alkaline phosphatase (ALP) activity. in vivo experiments were evaluated through a micro-computerized tomography (µ-CT) and histological assays. The analysis confirmed that an increase in the concentration of the GDD in the sponge leads to a higher bone formation and deposition in rat calvarial defects. Histological assay results were in line with µ-CT. The results reported in this study demonstrated the potential application of GDD sponges as osteoinductor in bone tissue engineering in pathological or nonunion bone defects.

Methylcobalamin-Loaded PLCL Conduits Facilitate the Peripheral Nerve Regeneration.

The feasible fabrication of nerve guidance conduits (NGCs) with good biological performance is important for translation in clinics. In this study, poly(d,l-lactide-co-caprolactone) (PLCL) films loaded with various amounts (wt; 5%, 15%, 25%) of methylcobalamin (MeCbl) are prepared, and are further rolled and sutured to obtain MeCbl-loaded NGCs. The MeCbl can be released in a sustainable manner up to 21 days. The proliferation and elongation of Schwann cells, and the proliferation of Neuro2a cells are enhanced on these MeCbl-loaded films. The MeCbl-loaded NGCs are implanted into rats to induce the regeneration of 10 mm amputated sciatic nerve defects, showing the ability to facilitate the recovery of motor and sensory function, and to promote myelination in peripheral nerve regeneration. In particular, the 15% MeCbl-loaded PLCL conduit exhibits the most satisfactory recovery of sciatic nerves in rats with the largest diameter and thickest myelinated fibers.

Tubular scaffold with microchannels and an H-shaped lumen loaded with bone marrow stromal cells promotes neuroregeneration and inhibits apoptosis after spinal cord injury.

As a result of its complex histological structure, regeneration patterns of grey and white matter are quite different in the spinal cord. Therefore, tissue engineering scaffolds for repairing spinal cord injury must be able to adapt to varying neural regeneration patterns. The aim of the present study was to improve a previously reported spinal cord-mimicking partition-type scaffold by adding microchannels on a single tubular wall along its longitudinal axis, thus integrating the two architectures of a single H-shaped central tube and many microchannels. Next, the integrated scaffold was loaded with bone marrow stromal cells (BMSCs) and transplanted to bridge the 5-mm defect of a complete transverse lesion in the thoracic spinal cord of rats. Subsequently, effects on nerve regeneration, locomotion function recovery, and early neuroprotection were observed. After 1 year of repair, the integrated scaffold could guide the regeneration of axons appearing in the debris of degraded microchannels, especially serotonin receptor 1A receptor-positive axonal tracts, which were relatively orderly arranged. Moreover, a network of nerve fibres was present, and a few BMSCs expressed neuronal markers in tubular lumens. Functionally, electrophysiological and locomotor functions of rats were partially recovered. In addition, we found that BMSCs could protect neurons and oligodendrocytes from apoptosis during the early stage of implantation. Taken together, our results demonstrate the potential of this novel integrated scaffold loaded with BMSCs to promote spinal cord regeneration through mechanical guidance and neuroprotective mechanisms.

Repair of lumbar vertebral bone defects by bone particles combined with hUC-MSCs in weaned rabbit.

Aim: The major symptom of many closed spinal dysraphism patients is that the laminas or arches of vertebra are not fused well. To date, the bone repair of spina bifida for young children is a significant challenge in clinical practice. Materials & methods: Bovine bone collagen particle (BBCP) scaffolds combined with human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were implanted in the defect area. X-ray analysis was performed after 3 months. Tissues were harvested for gross observation, and histological and immunohistochemical staining. Results: The BBCP supported hUC-MSCs adhesion and growth. Implanted BBCP combined with hUC-MSCs also promoted bone regeneration in the vertebral lamina and arch defect area. Conclusion: This method represents a new strategy for vertebral lamina and arch reconstruction in children.

Biological evaluation of porous nanocomposite scaffolds based on strontium substituted ß-TCP and bioactive glass: An in vitro and in vivo study.

In the current study, in vitro analysis of the osteogenic potential of different scaffolds based on strontium-substituted ß-TCP (Sr-TCP) and bioactive glass (BG) ceramics was conducted using rabbit bone marrow-derived mesenchymal stem cells (rBMSCs) and the osteogenic ability of the prepared Sr-TCP and BG scaffold was evaluated through alkaline phosphatase activity, mineral deposition by Alizarin red staining, and osteoblastic gene expression experiments. The obtained in vitro results revealed that among experimental Sr-TCP/BG nanocomposite scaffold samples with the composition of Sr-TCP/BG: 100/0, 50/50, 75/25, and 25/75, the 50Sr-TCP/50BG sample presented better osteoinductive properties. Therefore, the optimized 50Sr-TCP/50BG nanocomposite scaffold was chosen for further in vivo experiments. In vivo implantation of 50Sr-TCP/50BG scaffold and hydroxyapatite (HA)/TCP granules in a rabbit calvarial defect model showed slow degradation of 50Sr-TCP/50BG scaffold and high resorption rate of HA/TCP granules at 5 months' post-surgery. However, the 50Sr-TCP/50BG scaffolds loaded by mesenchymal stem cells (MSCs) were mainly replaced with new bone even at 2 months post-implantation. Based on the obtained engineering and biological results, 50Sr-TCP/50BG nanocomposite scaffold containing MSCs could be considered as a promising alternative substitute even for load-bearing bone tissue engineering applications.

3D bioprinted endometrial stem cells on melt electrospun poly ε-caprolactone mesh for pelvic floor application promote anti-inflammatory responses in mice.

Endometrial mesenchymal stem/stromal cells (eMSCs) exhibit excellent regenerative capacity in the endometrial lining of the uterus following menstruation and high proliferative capacity in vitro. Bioprinting eMSCs onto a mesh could be a potential therapy for Pelvic Organ Prolapse (POP). This study reports an alternative treatment strategy targeting vaginal wall repair using bioprinting of eMSCs encapsulated in a hydrogel and 3D melt electrospun mesh to generate a tissue engineering construct. Following a CAD, 3D printed poly ε-caprolactone (PCL) meshes were fabricated using melt electrospinning (MES) at different temperatures using a GMP clinical grade GESIM Bioscaffolder. Electron and atomic force microscopies revealed that MES meshes fabricated at 100 °C and with a speed 20 mm/s had the largest open pore diameter (47.2 ±â€¯11.4 µm) and the lowest strand thickness (121.4 ±â€¯46 µm) that promoted optimal eMSC attachment. An Aloe Vera-Sodium Alginate (AV-ALG) composite based hydrogel was optimised to a 1:1 mixture (1%AV-1%ALG) and eMSCs, purified from human endometrial biopsies, were then bioprinted in this hydrogel onto the MES printed meshes. Acute in vivo foreign body response assessment in NSG mice revealed that eMSC printed on MES constructs promoted tissue integration, eMSC retention and an anti-inflammatory M2 macrophage phenotype characterised by F4/80+CD206+ colocalization. Our results address an unmet medical need highlighting the potential of 3D bioprinted eMSC-MES meshes as an alternative approach to overcome the current challenges with non-degradable knitted meshes in POP treatment. STATEMENT OF SIGNIFICANCE: This study presents the first report of bioprinting mesenchymal stem cells derived from woman endometrium (eMSCs) to boost Pelvic Organ Prolapse (POP) treatment. It impacts over 50% of elderly women with no optimal treatment at present. The overall study is conducted in three stages as fabricating a melt electrospun (MES) mesh, bioprinting eMSCs into a Ca2+ free Aloe Vera-Alginate (AV-Alg) based hydrogel and in vivo study. Our data showed that AV-ALG hydrogel potentially suppresses the foreign body response and further addition of eMSCs triggered a high influx of anti-inflammatory CD206+ M2 macrophages. Our final construct demonstrates a favourable foreign body response to predict expected tissue integration, therefore, provides a potential for developing an alternative treatment for POP.

NK-Cell-Encapsulated Porous Microspheres via Microfluidic Electrospray for Tumor Immunotherapy.

Immunotherapy has recently garnered significant research interest in the field of clinical cancer management. The potential of tumor immunotherapy has been demonstrated for targeting a variety of tumors, both in vivo and in vitro, yielding some remarkable therapeutic effects. Herein, inspired by the stem cell niche, we developed a scale-up approach to generating porous microspheres with encapsulated natural killer (NK) cells via microfluidic electrospray for in situ tumor immunotherapy. The generated microspheres contained porous microstructures with tunable morphologies because of versatile but precise fluid control in the microfluidic electrospray system. NK-92MI cells encapsulated in porous microspheres were protected from the outer complex environment, allowing for improved proliferation and functionality. As observed, perforin and granzymes were sustainably secreted from the encapsulated NK-92MI cells, which exhibited robust killing effects on tumors both in vitro and in vivo. With continual proliferation, NK-92MI cells budded from the surface of porous microspheres and migrated into the surrounding residual tumor tissues, further destroying tumor cells. More importantly, no side effects owing to the native host immune system were observed by injecting the NK-92MI cell-encapsulated microspheres into tumors in vivo. Therefore, the NK-cell-encapsulated porous microspheres show great potential for tumor immunotherapy, offering a robust and attractive treatment option for cancer patient management.

Cardiac regeneration using human-induced pluripotent stem cell-derived biomaterial-free 3D-bioprinted cardiac patch in vivo.

One of the leading causes of death worldwide is heart failure. Despite advances in the treatment and prevention of heart failure, the number of affected patients continues to increase. We have recently developed 3D-bioprinted biomaterial-free cardiac tissue that has the potential to improve cardiac function. This study aims to evaluate the in vivo regenerative potential of these 3D-bioprinted cardiac patches. The cardiac patches were generated using 3D-bioprinting technology in conjunction with cellular spheroids created from a coculture of human-induced pluripotent stem cell-derived cardiomyocytes, fibroblasts, and endothelial cells. Once printed and cultured, the cardiac patches were implanted into a rat myocardial infarction model (n = 6). A control group (n = 6) without the implantation of cardiac tissue patches was used for comparison. The potential for regeneration was measured 4 weeks after the surgery with histology and echocardiography. 4 weeks after surgery, the survival rates were 100% and 83% in the experimental and the control group, respectively. In the cardiac patch group, the average vessel counts within the infarcted area were higher than those within the control group. The scar area in the cardiac patch group was significantly smaller than that in the control group. (Figure S1) Echocardiography showed a trend of improvement of cardiac function for the experimental group, and this trend correlated with increased patch production of extracellular vesicles. 3D-bioprinted cardiac patches have the potential to improve the regeneration of cardiac tissue and promote angiogenesis in the infarcted tissues and reduce the scar tissue formation.

Evaluation of cartilage regeneration of chondrocyte encapsulated gellan gum-based hyaluronic acid blended hydrogel.

Hydrogels have shown to be advantageous in supporting damaged cartilage because of its analogous to the extracellular matrix (ECM) of cartilage tissue. However, problems such as infection and inflammation are still a challenge to be solved. In terms of tissue engineering, natural materials are more advantageous than synthetic materials in biocompatibility and biodegradability status. Herein, physically blended nature-derived gellan gum (GG) hydrogel and hyaluronic acid (HA) hydrogel is suggested as a one of solution for cartilage tissue engineering material. The purpose of this study is to determine the effect of GG/HA hydrogel in vitro and in vivo. The chemical and mechanical properties were measured to confirm the compatibility of hydrogels for cartilage tissue engineering. The viability, proliferation, morphology, and gene expression of chondrocytes encapsulated in hydrogels were examined in vitro. Furthermore, the beneficial effect of the blended hydrogel was confirmed by performing the in vivo experiment. The chemical properties of hydrogels confirmed the well physically blended hydrogels. The mechanical studies of hydrogels displayed that as the content of HA increases, the swelling ratio was higher, compressive strength decreased and degradation was faster. Therefore, to use the hydrogel of GG and HA network, the proper amount must be blended. The in vitro study of chondrocytes encapsulated GG/HA hydrogel showed that the proper amount of HA enhanced the cell growth, attachment, and gene expression. The in vivo examination verified the advantageous effect of GG/HA hydrogel. Overall results demonstrate that GG/HA hydrogel is suitable for culturing chondrocyte and can be further applied for the treatment of cartilage defects.

Electrospun Nanofiber-Based Patches for the Delivery of Cardiac Progenitor Cells.

Congenital heart disease is the number one cause of birth defect-related death because it often leads to right ventricular heart failure (RVHF). One promising avenue to combat this RVHF is the use of cardiac patches composed of stem cells and scaffolds. Herein, we demonstrate a reparative cardiac patch by combining neonatal or child c-kit+  progenitor cells (CPCs) with a scaffold composed of electrospun polycaprolactone nanofibers. We examined different parameters of the patch, including the alignment, composition, and surface properties of the nanofibers, as well as the age of the CPCs. The patch based on uniaxially aligned nanofibers successfully aligned the CPCs. With the inclusion of gelatin in the nanofiber matrix and/or coating of fibronectin on the surface of the nanofibers, the metabolism of both neonatal and child CPCs was generally enhanced. The conditioned media collected from both patches based on aligned and random nanofibers could reduce the fibrotic gene expression in rat cardiac fibroblasts, following stimulation with transforming growth factor ß. Furthermore, the conditioned media collected from the nanofiber-based patches could lead to the formation of tubes of human umbilical vein endothelial cells, indicating the pro-angiogenic capability of the patch. Taken together, the electrospun nanofiber-based patches are a suitable delivery vehicle for CPCs and can confer reparative benefit through anti-fibrotic and pro-angiogenic paracrine signaling.

Multifunctionalized hydrogels foster hNSC maturation in 3D cultures and neural regeneration in spinal cord injuries.

Three-dimensional cell cultures are leading the way to the fabrication of tissue-like constructs useful to developmental biology and pharmaceutical screenings. However, their reproducibility and translational potential have been limited by biomaterial and culture media compositions, as well as cellular sources. We developed a construct comprising synthetic multifunctionalized hydrogels, serum-free media, and densely seeded good manufacturing practice protocol-grade human neural stem cells (hNSC). We tracked hNSC proliferation, differentiation, and maturation into GABAergic, glutamatergic, and cholinergic neurons, showing entangled electrically active neural networks. The neuroregenerative potential of the "engineered tissue" was assessed in spinal cord injuries, where hNSC-derived progenitors and predifferentiated hNSC progeny, embedded in multifunctionalized hydrogels, were implanted. All implants decreased astrogliosis and lowered the immune response, but scaffolds with predifferentiated hNSCs showed higher percentages of neuronal markers, better hNSC engraftment, and improved behavioral recovery. Our hNSC-construct enables the formation of 3D functional neuronal networks in vitro, allowing novel strategies for hNSC therapies in vivo.

Adipose-derived stem cells promote survival, growth, and maturation of early-stage murine follicles.

BACKGROUND: Premature ovarian insufficiency is a common complication of anticancer treatments in young women and girls. The ovary is a complex, highly regulated reproductive organ, whose proper function is contingent upon the bidirectional endocrine, paracrine, and autocrine signaling. These factors facilitate the development of the follicles, the functional units of the ovary, to progress from the gonadotropin-independent, paracrine-controlled early stage to the gonadotropin-dependent, endocrine-controlled later stage. We hypothesized that the low survival rate of individually cultured early-stage follicles could be improved with co-culture of adipose-derived stem cells (ADSCs) that secrete survival- and growth-promoting factors. MATERIALS AND METHODS: Ovarian follicles ranging from 85 to 115 µm in diameter, from 10- to 12-day-old B6CBAF1 mice were mechanically isolated and co-encapsulated with ADSCs within alginate-based 3D culture system. The follicles were cultured for 14 days, imaged using light microscopy every 2 days, and matured at the end. Follicle media were changed every 2 days and collected for hormone measurements. Follicle diameter, morphology, number of transzonal projections, and survival and maturation rates were recorded. Statistical analyses using one- and two-way ANOVA were performed to compare hormone levels, survival of the follicles and ADSCs, oocyte maturation rates, and follicle growth. RESULTS: The co-encapsulation of the follicles with ADSCs increased follicle survival, ranging from 42.4% for the 86-95 µm to 86.2% for the 106-115-µm follicle size group. Co-culture also improved the follicle growth, the rate of antrum formation and oocyte maturation compared to the follicles cultured alone. The levels of androstenedione, estradiol, and progesterone of co-encapsulated follicles increased progressively with time in culture. CONCLUSIONS: To our knowledge, this is the first report of an in vitro system utilizing mouse adipose-derived stem cells to support the development of the mouse follicles. Our findings suggest that co-encapsulation of ADSCs with early-stage follicles supports follicular development, through secretion of cytokines that promote follicular survival, antrum formation, and meiotic competence. The unique 3D culture system that supports the survival of both cell types has translational implications, as ADSCs could be used as an autologous source for in vitro maturation of early-stage human follicles.

3D-3 Tumor Models in Drug Discovery for Analysis of Immune Cell Infiltration.

The cross talk between tumor cells and other cells present in the tumor microenvironment such as stromal and immune cells highly influences the behavior and progression of disease. Understanding the underlying mechanisms of interaction is a prerequisite to develop new treatment strategies and to prevent or at least reduce therapy failure in the future. Specific reactivation of the patient's immune system is one of the major goals today. However, standard two-dimensional (2D) cell culture techniques lack the necessary complexity to address related questions. Novel three-dimensional (3D) in vitro models-embedded in a matrix or encapsulated in alginate-recapitulate the in vivo situation much better. Cross talk between different cell types can be studied starting from co-cultures. As cancer immune modulation is becoming a major research topic, 3D in vitro models represent an important tool to address immune regulatory/modulatory questions for T, NK, and other cells of the immune system. The 3D systems consisting of tumor cells, fibroblasts, and immune cells (3D-3) already proved as a reliable tool for us. For instance, we made use of those models to study the molecular mechanisms of the cross talk of non-small cell lung cancer (NSCLC) and fibroblasts, to unveil macrophage plasticity in the tumor microenvironment and to mirror drug responses in vivo. Generation of those 3D models and how to use them to study immune cell infiltration and activation will be described in the present book chapter.

Osteogenic Effects of VEGF-Overexpressed Human Adipose-Derived Stem Cells with Whitlockite Reinforced Cryogel for Bone Regeneration.

Bone is a vascularized tissue that is comprised of collagen fibers and calcium phosphate crystals such as hydroxyapatite (HAp) and whitlockite (WH). HAp and WH are known to elicit bone regeneration by stimulating osteoblast activities and osteogenic commitment of stem cells. In addition, vascular endothelial growth factor (VEGF) is shown to promote osteogenesis and angiogenesis which is considered as an essential process in bone repair by providing nutrients. In this study, VEGF-secreting human adipose-derived stem cells (VEGF-ADSCs) are developed by transducing ADSCs with VEGF-encoded lentivirus. Additionally, WH-reinforced gelatin/heparin cryogels (WH-C) are fabricated by loading WH into gelatin/heparin cryogels. VEGF-ADSC secrete tenfold more VEGF than ADSC and show increased VEGF secretion with cell growth. Also, incorporation of WH into cryogels provides a mineralized environment with ions secreted from WH. When the VEGF-ADSCs are seeded on WH-C, sustained release of VEGF is observed due to the specific affinity of VEGF to heparin. Finally, the synergistic effect of VEGF-ADSC and WH on osteogenesis is successfully confirmed by alkaline phosphatase and real-time polymerase chain reaction analysis. In vivo bone formation is demonstrated via implantation of VEGF-ADSC seeded WH-C into mouse calvarial bone defect model, resulted in enhanced bone development with the highest bone volume/total volume.

Combinatorial tissue engineering partially restores function after spinal cord injury.

Hydrogel scaffolds provide a beneficial microenvironment in transected rat spinal cord. A combinatorial biomaterials-based strategy provided a microenvironment that facilitated regeneration while reducing foreign body reaction to the three-dimensional spinal cord construct. We used poly lactic-co-glycolic acid microspheres to provide sustained release of rapamycin from Schwann cell (SC)-loaded, positively charged oligo-polyethylene glycol fumarate scaffolds. The biological activity and dose-release characteristics of rapamycin from microspheres alone and from microspheres embedded in the scaffold were determined in vitro. Three dose formulations of rapamycin were compared with controls in 53 rats. We observed a dose-dependent reduction in the fibrotic reaction to the scaffold and improved functional recovery over 6 weeks. Recovery was replicated in a second cohort of 28 animals that included retransection injury. Immunohistochemical and stereological analysis demonstrated that blood vessel number, surface area, vessel diameter, basement membrane collagen, and microvessel phenotype within the regenerated tissue was dependent on the presence of SCs and rapamycin. TRITC-dextran injection demonstrated enhanced perfusion into scaffold channels. Rapamycin also increased the number of descending regenerated axons, as assessed by Fast Blue retrograde axonal tracing. These results demonstrate that normalization of the neovasculature was associated with enhanced axonal regeneration and improved function after spinal cord transection.