Vascularization of cutaneous wounds by stem cells.

Differentiated skin cells have limited self-renewal capacity; thus, the application of stem/progenitor cells, adult or induced stem cells, has attracted much attention for wound healing applications. Upon skin injury, vascularization, known as a highly dynamic process, occurs with the contribution of cells, the extracellular matrix, and relevant growth factors. Considering the importance of this process in tissue regeneration, several strategies have been proposed to enhance angiogenesis and accelerate wound healing. Previous studies report the effectiveness of stem/progenitor cells in skin wound healing by facilitating the vascularization process. This chapter reviews and highlights some of the key and recent investigations on application of stem/progenitor cells to induce skin revascularization after trauma.

Exosome engineering in cell therapy and drug delivery.

Cell-derived exosomes have opened new horizons in modern therapy for advanced drug delivery and therapeutic applications, due to their key features such as low immunogenicity, high physicochemical stability, capacity to penetrate into tissues, and the innate capacity to communicate with other cells over long distances. Exosome-based liquid biopsy has been potentially used for the diagnosis and prognosis of a range of disorders. Exosomes deliver therapeutic agents, including immunological modulators, therapeutic drugs, and antisense oligonucleotides to certain targets, and can be used as vaccines, though their clinical application is still far from reality. Producing exosomes on a large-scale is restricted to their low circulation lifetime, weak targeting capacity, and inappropriate controls, which need to be refined before being implemented in practice. Several bioengineering methods have been used for refining therapeutic applications of exosomes and promoting their effectiveness, on the one hand, and addressing the existing challenges, on the other. In the short run, new diagnostic platforms and emerging therapeutic strategies will further develop exosome engineering and therapeutic potential. This requires a thorough analysis of exosome engineering approaches along with their merits and drawbacks, as outlined in this paper. The present study is a comprehensive review of novel techniques for exosome development in terms of circulation time in the body, targeting capacity, and higher drug loading/delivery efficacies.

Convergence of Biofabrication Technologies and Cell Therapies for Wound Healing.

BACKGROUND: Cell therapy holds great promise for cutaneous wound treatment but presents practical and clinical challenges, mainly related to the lack of a supportive and inductive microenvironment for cells after transplantation. Main: This review delineates the challenges and opportunities in cell therapies for acute and chronic wounds and highlights the contribution of biofabricated matrices to skin reconstruction. The complexity of the wound healing process necessitates the development of matrices with properties comparable to the extracellular matrix in the skin for their structure and composition. Over recent years, emerging biofabrication technologies have shown a capacity for creating complex matrices. In cell therapy, multifunctional material-based matrices have benefits in enhancing cell retention and survival, reducing healing time, and preventing infection and cell transplant rejection. Additionally, they can improve the efficacy of cell therapy, owing to their potential to modulate cell behaviors and regulate spatiotemporal patterns of wound healing. CONCLUSION: The ongoing development of biofabrication technologies promises to deliver material-based matrices that are rich in supportive, phenotype patterning cell niches and are robust enough to provide physical protection for the cells during implantation.

High-yield isolation of pure fetal endothelial colony forming cells and mesenchymal stem cells from the human full-term placenta.

A need to identify a stem cell source for human endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) that is high yield is crucial for their implementation in ischemia. Our lab has developed an isolation protocol to do this using full-term human villous placental tissue. This protocol describes enzymatic tissue digestion followed by MACS and FACS, achieving an 8 times greater yield versus traditional isolation techniques and delivering pure fetal stem cell colonies within 21-28 days cell culture. For complete details on the use and execution of this protocol, please refer to Patel et al. (2013) and Patel et al. (2014).

Strategies to Induce Blood Vessel Ingrowth into Skin Grafts and Tissue-Engineered Substitutes.

Skin is a multilayer organ consisting of several tissues and appendages residing in a complex niche. Adequate and physiologically regulated vascularization is an absolute requirement for skin homeostasis, regeneration, and wound healing. The lack of vascular networks and ischemia results in delayed wound closure. In addition, vascularization is critical for the prolonged function and survival of skin grafts and tissue-engineered skin substitutes. This study highlights the clinical challenges associated with the limited vascularization in the cutaneous wounds. Then, we highlight the novel approaches for the development of vascular networks in the skin autografts, allografts, and artificial substitutes. Also, the future directions to overcome the existing vascularization complications in skin grafting and synthetic skin substitutes are presented. Statement of Significance Delayed closure of large dermal wounds, such as burn injuries, results from the lack of vascular networks and ischemia. The amount of blood supply in the skin graft is the primary factor determining the quality of the transplanted grafts. The current skin grafts and their fabrication methods lack the appropriate features that contribute to the vascularization and integration of the wound bed and graft and adherence to the skin layers. Therefore, the new generation of skin grafts should consider advanced technologies to induce vascularization and overcome current challenges.

A humanised rat model of osteosarcoma reveals ultrastructural differences between bone and mineralised tumour tissue.

Current xenograft animal models fail to accurately replicate the complexity of human bone disease. To gain translatable and clinically valuable data from animal models, new in vivo models need to be developed that mimic pivotal aspects of human bone physiology as well as its diseased state. Above all, an advanced bone disease model should promote the development of new treatment strategies and facilitate the conduction of common clinical interventional procedures. Here we describe the development and characterisation of an orthotopic humanised tissue-engineered osteosarcoma (OS) model in a recently genetically engineered x-linked severe combined immunodeficient (X-SCID) rat. For the first time in a genetically modified rat, our results show the successful implementation of an orthotopic humanised tissue-engineered bone niche supporting the growth of a human OS cell line including its metastatic spread to the lung. Moreover, we studied the inter- and intraspecies differences in ultrastructural composition of bone and calcified tissue produced by the tumour, pointing to the crucial role of humanised animal models.

A humanized orthotopic tumor microenvironment alters the bone metastatic tropism of prostate cancer cells.

Prostate cancer (PCa) is the second most commonly diagnosed cancer in men, and bone is the most frequent site of metastasis. The tumor microenvironment (TME) impacts tumor growth and metastasis, yet the role of the TME in PCa metastasis to bone is not fully understood. We used a tissue-engineered xenograft approach in NOD-scid IL2Rγnull (NSG) mice to incorporate two levels of humanization; the primary tumor and TME, and the secondary metastatic bone organ. Bioluminescent imaging, histology, and immunohistochemistry were used to study metastasis of human PC-3 and LNCaP PCa cells from the prostate to tissue-engineered bone. Here we show pre-seeding scaffolds with human osteoblasts increases the human cellular and extracellular matrix content of bone constructs, compared to unseeded scaffolds. The humanized prostate TME showed a trend to decrease metastasis of PC-3 PCa cells to the tissue-engineered bone, but did not affect the metastatic potential of PCa cells to the endogenous murine bones or organs. On the other hand, the humanized TME enhanced LNCaP tumor growth and metastasis to humanized and murine bone. Together this demonstrates the importance of the TME in PCa bone tropism, although further investigations are needed to delineate specific roles of the TME components in this context.

Immunomodulatory and Therapeutic Effects of Mesenchymal Stem Cells on Organ Dysfunction in Sepsis.

ABSTRACT: Sepsis is a life-threatening disorder that is caused by a dysregulated inflammatory response during an infection. The disease mostly affects pregnant women, newborns, and patients in intensive care units. Sepsis treatment is a significant part of a country's health budgets. Delay in the therapy causes irreversible failure of various organs due to the lack of blood supply and reduction of oxygen in the tissues and eventually increased mortality. The involvement of four or five organs by sepsis has been attributed to an increased risk of death to over 90%. Although antibiotics are at the first line of sepsis treatment, they do not possess enough potency to control the disease and prevent subsequent organ failure. The immunomodulatory, anti-inflammatory, anti-apoptotic, and anti-microbial properties of mesenchymal stem cells (MSCs) have been reported in various studies. Therefore, the application of MSCs has been considered a potentially promising therapeutic strategy. In preclinical studies, the administration of MSCs has been associated with reduced bacterial load and decreased levels of pro-inflammatory factors as well as the improved function of the different vital organs, including heart, kidney, liver, and lungs. The current study provides a brief review of sepsis and its pathophysiology, and then highlights recent findings in the therapeutic effects of MSCs and MSC-derived secretome in improving sepsis-induced organ dysfunction. Besides, eligible sepsis candidates for MSC-therapy and the latest clinical findings in these areas have been reviewed.

Synergistic therapeutic effect of mesenchymal stem cells and tolerogenic dendritic cells in an acute colitis mouse model.

Cell-based therapy with tolerizing cells has been applied for the treatment of inflammatory bowel disease (IBD) in previous experimental and clinical studies with promising results. In the current study, we utilized the dextran sulfate sodium (DSS)-induced colitis model, to investigate if tolerogenic dendritic cell-mesenchymal stem cell (tDC-MSC) combination therapy can augment the therapeutic effects of single transplantation of each cell type. The effect of MSC and tDC co-transplantation on the severity of colitis was assessed by daily monitoring of body weight, stool consistency, and rectal bleeding, and compared with control groups. Moreover, the colon length, colon weight, myeloperoxidase (MPO) activity were measured and evaluated with histological analysis of colon tissues. The Treg cell percentage and cytokine levels in spleens and mesenteric lymph nodes (MLNs) were measured by flow cytometry and ELISA, respectively. The results showed co-transplantation of MSCs and tDCs was more effective in alleviating the clinical and histological manifestations of colitis than monotherapy, especially when compared with MSC alone. The protective effects of tDC-MSC were accompanied by the induction of Treg cells and increased the production of anti-inflammatory cytokines in spleens and mesenteric lymph nodes. Together, co-transplantation of MSCs and tDCs could be a promising and effective therapeutic approach in the treatment of IBD.

A 3D-printed biomaterials-based platform to advance established therapy avenues against primary bone cancers.

In this study we developed and validated a 3D-printed drug delivery system (3DPDDS) to 1) improve local treatment efficacy of commonly applied chemotherapeutic agents in bone cancers to ultimately decrease their systemic side effects and 2) explore its concomitant diagnostic potential. Thus, we locally applied 3D-printed medical-grade polycaprolactone (mPCL) scaffolds loaded with Doxorubicin (DOX) and measured its effect in a humanized primary bone cancer model. A bioengineered species-sensitive orthotopic humanized bone niche was established at the femur of NOD-SCID IL2Rγnull (NSG) mice. After 6 weeks of in vivo maturation into a humanized ossicle, Luc-SAOS-2 cells were injected orthotopically to induce local growth of osteosarcoma (OS). After 16 weeks of OS development, a biopsy-like defect was created within the tumor tissue to locally implant the 3DPDDS with 3 different DOX loading doses into the defect zone. Histo- and morphological analysis demonstrated a typical invasive OS growth pattern inside a functionally intact humanized ossicle as well as metastatic spread to the murine lung parenchyma. Analysis of the 3DPDDS revealed the implants' ability to inhibit tumor infiltration and showed local tumor cell death adjacent to the scaffolds without any systemic side effects. Together these results indicate a therapeutic and diagnostic capacity of 3DPDDS in an orthotopic humanized OS tumor model.

Mesenchymal stem cell therapies for COVID-19: Current status and mechanism of action.

The outbreak of COVID-19 in December 2019, has become an urgent and serious public health emergency. At present, there is no effective treatment or vaccine for COVID-19. Therefore, there is a crucial unmet need to develop a safe and effective treatment for COVID-19 patients. Mesenchymal stem cells (MSCs) are widely used in basic science and in a variety of clinical trials. MSCs are able to engraft to the damaged tissues after transplantation and promote tissue regeneration, besides MSCs able to secrete immunomodulatory factors that suppress the cytokine storms. Moreover, the contribution of MSCs to prevent cell death and inhibit tissue fibrosis is well established. In the current review article, the potential mechanisms by which MSCs contribute to the treatment of COVID-19 patients are highlighted. Also, current trials that evaluated the potential of MSC-based treatments for COVID-19 are briefly reviewed.

Engineering a Humanised Niche to Support Human Haematopoiesis in Mice: Novel Opportunities in Modelling Cancer.

Despite the bone marrow microenvironment being widely recognised as a key player in cancer research, the current animal models that represent a human haematopoietic system lack the contribution of the humanised marrow microenvironment. Here we describe a murine model that relies on the combination of an orthotopic humanised tissue-engineered bone construct (ohTEBC) with patient-specific bone marrow (BM) cells to create a humanised bone marrow (hBM) niche capable of supporting the engraftment of human haematopoietic cells. Results showed that this model supports the engraftment of human CD34+ cells from a healthy BM with human haematopoietic cells migrating into the mouse BM, human BM compartment, spleen and peripheral blood. We compared these results with the engraftment capacity of human CD34+ cells obtained from patients with multiple myeloma (MM). We demonstrated that CD34+ cells derived from a diseased BM had a reduced engraftment potential compared to healthy patients and that a higher cell dose is required to achieve engraftment of human haematopoietic cells in peripheral blood. Finally, we observed that hematopoietic cells obtained from the mobilised peripheral blood of patients yields a higher number of CD34+, overcoming this problem. In conclusion, this humanised mouse model has potential as a unique and patient-specific pre-clinical platform for the study of tumour-microenvironment interactions, including human bone and haematopoietic cells, and could, in the future, serve as a drug testing platform.

Correction: Humanized bone facilitates prostate cancer metastasis and recapitulates therapeutic effects of Zoledronic acid in vivo.

[This corrects the article DOI: 10.1038/s41413-019-0072-9.].

Targeted camptothecin delivery via silicon nanoparticles reduces breast cancer metastasis.

In advanced breast cancer (BCa) patients, not the primary tumor, but the development of distant metastases, which occur mainly in the organ bone, and their adverse health effects are responsible for high mortality. Targeted delivery of already known drugs which displayed potency, but rather unfavorable pharmacokinetic properties, might be a promising approach to overcome the current limitations of metastatic BCa therapy. Camptothecin (CPT) is a highly cytotoxic chemotherapeutic compound, yet poorly water-soluble and non-specific. Here, CPT was loaded into porous silicon nanoparticles (pSiNP) displaying the epidermal growth factor receptor (EGFR)-targeting antibody (Ab) cetuximab to generate a soluble and targeted nanoscale delivery vehicle for cancer treatment. After confirming the cytotoxic effect of targeted CPT-loaded pSiNP in vitro on MDA-MB-231BO cells, nanoparticles were studied in a humanized BCa bone metastasis mouse model. Humanized tissue-engineered bone constructs (hTEBCs) provided a humanized microenvironment for BCa bone metastases in female NOD-scid IL2Rgnull (NSG) mice. Actively targeted CPT-loaded pSiNP led to a reduction of orthotopic primary tumor growth, increased survival rate and significant decrease in hTEBC and murine lung, liver and bone metastases. This study demonstrates that targeted delivery via pSiNP is an effective approach to employ CPT and other potent anti-cancer compounds with poor pharmacokinetic profiles in cancer therapy.

Design and Fabrication of Three-Dimensional Printed Scaffolds for Cancer Precision Medicine.

Three-dimensional (3D)-engineered scaffolds have been widely investigated as drug delivery systems (DDS) or cancer models with the aim to develop effective cancer therapies. The in vitro and in vivo models developed via 3D printing (3DP) and tissue engineering concepts have significantly contributed to our understanding of cell-cell and cell-extracellular matrix interactions in the cancer microenvironment. Moreover, 3D tumor models were used to study the therapeutic efficiency of anticancer drugs. The present study aims to provide an overview of applying the 3DP and tissue engineering concepts for cancer studies with suggestions for future research directions. The 3DP technologies being used for the fabrication of personalized DDS have been highlighted and the potential technical approaches and challenges associated with the fused deposition modeling, the inkjet-powder bed, and stereolithography as the most promising 3DP techniques for drug delivery purposes are briefly described. Then, the advances, challenges, and future perspectives in tissue-engineered cancer models for precision medicine are discussed. Overall, future advances in this arena depend on the continuous integration of knowledge from cancer biology, biofabrication techniques, multiomics and patient data, and medical needs to develop effective treatments ultimately leading to improved clinical outcomes. Impact statement Three-dimensional printing (3DP) enables the fabrication of personalized medicines and drug delivery systems. The convergence of 3DP, tissue engineering concepts, and cancer biology could significantly improve our understanding of cancer biology and contribute to the development of new cancer therapies.

Humanized bone facilitates prostate cancer metastasis and recapitulates therapeutic effects of zoledronic acid in vivo.

Advanced prostate cancer (PCa) is known for its high prevalence to metastasize to bone, at which point it is considered incurable. Despite significant effort, there is no animal model capable of recapitulating the complexity of PCa bone metastasis. The humanized mouse model for PCa bone metastasis used in this study aims to provide a platform for the assessment of new drugs by recapitulating the human-human cell interactions relevant for disease development and progression. The humanized tissue-engineered bone construct (hTEBC) was created within NOD-scid IL2rgnull (NSG) mice and was used for the study of experimental PC3-Luc bone metastases. It was confirmed that PC3-Luc cells preferentially grew in the hTEBC compared with murine bone. The translational potential of the humanized mouse model for PCa bone metastasis was evaluated with two clinically approved osteoprotective therapies, the non-species-specific bisphosphonate zoledronic acid (ZA) or the human-specific antibody Denosumab, both targeting Receptor Activator of Nuclear Factor Kappa-Β Ligand. ZA, but not Denosumab, significantly decreased metastases in hTEBCs, but not murine femora. These results highlight the importance of humanized models for the preclinical research on PCa bone metastasis and indicate the potential of the bioengineered mouse model to closely mimic the metastatic cascade of PCa cells to human bone. Eventually, it will enable the development of new effective antimetastatic treatments.

Microenvironment engineering of osteoblastic bone metastases reveals osteomimicry of patient-derived prostate cancer xenografts.

Representative in vitro models that mimic the native bone tumor microenvironment are warranted to support the development of more successful treatments for bone metastases. Here, we have developed a primary cell 3D model consisting of a human osteoblast-derived tissue-engineered construct (hOTEC) indirectly co-cultured with patient-derived prostate cancer xenografts (PDXs), in order to study molecular interactions in a patient-derived microenvironment context. The engineered biomimetic microenvironment had high mineralization and embedded osteocytes, and supported a high degree of cancer cell osteomimicry at the gene, protein and mineralization levels when co-cultured with prostate cancer PDXs from a lymph node metastasis (LuCaP35) and bone metastasis (BM18) from patients with primary prostate cancer. This fully patient-derived model is a promising tool for the assessment of new molecular mechanisms and as a personalized pre-clinical platform for therapy testing for patients with prostate cancer bone metastases.

Recombinant Human Bone Morphogenetic Protein 7 Exerts Osteo-Catabolic Effects on Bone Grafts That Outweigh Its Osteo-Anabolic Capacity.

This study aimed to investigate the effects of recombinant human bone morphogenetic protein (rhBMP-7) on human cancellous bone grafts (BGs) while differentiating between anabolic and catabolic events. Human BGs alone or supplemented with rhBMP-7 were harvested 14 weeks after subcutaneous implantation into NOD/Scid mice, and studied via micro-CT, histomorphometry, immunohistochemistry and flow cytometry. Immunohistochemical staining for human-specific proteins made it possible to differentiate between grafted human bone and newly formed murine bone. Only BGs implanted with rhBMP-7 formed an ossicle containing a functional hematopoietic compartment. The total ossicle volume in the BMP+ group was higher than in the BMP- group (835 mm3 vs. 365 mm3, respectively, p < 0.001). The BMP+ group showed larger BM spaces (0.47 mm vs. 0.28 mm, p = 0.002) and lower bone volume-to-total volume ratio (31% vs. 47%, p = 0.002). Immunohistochemical staining for human-specific proteins confirmed a higher ratio of newly formed bone area (murine) to total area (0.12 vs. 0.001, p < 0.001) in the BMP+ group, while the ratio of grafted bone (human) area to total area was smaller (0.14 vs. 0.34, p = 0.004). The results demonstrate that rhBMP-7 induces BG resorption at a higher rate than new bone formation while creating a haematopoietic niche. Clinicians therefore need to consider the net catabolic effect when rhBMP-7 is used with BGs. Overall, this model indicates its promising application to further decipher BMPs action on BGs and its potential in complex bone tissue regeneration.

3D printed dual macro-, microscale porous network as a tissue engineering scaffold with drug delivering function.

Tissue engineering macroporous scaffolds are important for regeneration of large volume defects resulting from diseases such as breast or bone cancers. Another important part of the treatment of these conditions is adjuvant drug therapy to prevent disease recurrence or surgical site infection. In this study, we developed a new type of macroporous scaffolds that have drug loading and release functionality to use in these scenarios. 3D printing allows for building macroporous scaffolds with deterministically designed complex architectures for tissue engineering yet they often have low surface areas thus limiting their drug loading capability. In this proof-of-concept study, we aimed to introduce microscale porosity into macroporous scaffolds to allow for efficient yet simple soak-loading of various clinical drugs and control their release. Manufacturing of scaffolds having both macroporosity and microscale porosity remains a difficult task. Here, we combined porogen leaching and 3D printing to achieve this goal. Porogen microparticles were mixed with medical grade polycaprolactone and extruded into scaffolds having macropores of 0.7 mm in size. After leaching, intra-strut microscale pores were realized with pore size of 20-70 µm and a total microscale porosity of nearly 40%. Doxorubicin (DOX), paclitaxel (PTX) and cefazolin (CEF) were chosen as model drugs of different charges and solubilities to soak-load the scaffolds and achieved loading efficiency of over 80%. The microscale porosity was found to significantly reduce the burst release allowing the microporous scaffolds to release drugs up to 200, 500 and 150 h for DOX, PTX and CEF, respectively. Finally, cell assays were used and confirmed the bioactivities and dose response of the drug-loaded scaffolds. Together, the findings from this proof-of-concept study demonstrate a new type of scaffolds with dual micro-, macro-porosity for tissue engineering applications with intrinsic capability for efficient loading and sustained release of drugs to prevent post-surgery complications.

Immunosuppression Agent Cyclosporine Reduces Self-Renewal and Vessel Regeneration Potentiation of Human Endothelial Colony Forming Cells.

Endothelial colony forming cells (ECFC) and mesenchymal stem cells (MSC) combined have great potential to be used for cell therapy of ischemic vascular diseases. However, to improve allogeneic stem cell engraftment the use of immunosuppression, such as cyclosporine has been suggested. Our aim was to assess the impact of cyclosporine on hind limb revascularisation upon MSC and ECFC combination therapy. Balb/c immunocompetent mice subjected to hind limb ischemia (right femoral artery ligation) were given both human ECFC and MSC (weekly intramuscular injections) with or without cyclosporine (daily injection). Surprisingly, mice receiving cyclosporine had a significant decrease in reperfusion based on laser Doppler imaging compared to vehicle controls and had poorer limb survival. In vitro, the downstream calcineurin target NFATC4 was highly expressed in the self-renewing fraction of ECFCs. ECFCs cultured with cyclosporine had reduced colony formation capacity and tube formation in Matrigel. Lastly, ECFC displayed increased proliferation and loss of capacity for long term culture when in the presence of cyclosporine clearly showing a loss of quiescence and progenitor function. Our findings demonstrate the deleterious impact of cyclosporine on ECFC function, with significant impact on ECFC-based allogeneic cellular therapy. Stem Cells Translational Medicine 2019;8:162&7.