ABSTRACT The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the for... more ABSTRACT The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the form of liposomal nanoparticles to treat osteosarcoma (OS). Curcumin is water insoluble and an effective delivery route is through encapsulation in cyclodextrins followed by a second encapsulation in liposomes. Liposomal curcumin's potential was evaluated against cancer models of mesenchymal (OS) and epithelial origin (breast cancer). The resulting 2-Hydroxypropyl-γ-cyclodextrin/curcumin - liposome complex shows promising anticancer potential both in vitro and in vivo against KHOS OS cell line and MCF-7 breast cancer cell line. An interesting aspect is that liposomal curcumin initiates the caspase cascade that leads to apoptotic cell death in vitro in comparison with DMSO-curcumin induced autophagic cell death. In addition, the efficiency of the liposomal curcumin formulation was confirmed in vivo using a xenograft OS model. Curcumin-loaded γ-cyclodextrin liposomes indicate significant potential as delivery vehicles for the treatment of cancers of different tissue origin. FROM THE CLINICAL EDITOR: Curcumin-loaded γ-cyclodextrin liposomes were demonstrated in vitro to have significant potential as delivery vehicles for the treatment of cancers of mesenchymal and epithelial origin. Differences between mechanisms of cell death were also evaluated.
Patient Derived Xenografts (PDXs) are valuable disease models as they preserve the tumor microenv... more Patient Derived Xenografts (PDXs) are valuable disease models as they preserve the tumor microenvironment’s structure, providing the most clinically relevant in vivo testing platform. Nevertheless, most of those models still use animal derived hydrogels as scaffolds for patient derived cells, reducing the potential to fully mimic the physiological microenvironment of the disease and human condition. ObaGel® is a human-derived hydrogel successfully deployed for supporting 3D cell culture, organoid, perfusion bioreactor, and/microphysiological system development for studying obesity, type II diabetes, and various forms of related cancers. Here we are investigating the use of Obagel® in the in-vivo development of a PDX-derived culture of lung adenocarcinoma and compare its performance to currently used animal derived hydrogels. Methods: 3 × 10^6 CRT00295 Non-small cell lung adenocarcinoma cells in 100ul 50:50 indicated Gel in PBS per injection site; the study used 10 NOG mice per condition. Results: The results indicated that ObaGel® performed similarly in terms of engraftment and tumor kinetics compared to animal derived hydrogels such as Cultrex, Geltrex, and Matrigel, using a patient-derived xenograft culture of lung adenocarcinoma. Conclusions: Our data supports the use of a human-derived hydrogel for PDX models, ObaGel® performs equivalently to animal derived hydrogels to support tumor volume over time. The use of a human derived hydrogels as scaffolds for PDX models provide a better humanized local microenvironment for lung adenocarcinoma. Obagel® can also be adapted for use as xenografts in other tissue/disease models Citation Format: Cecilia Sanchez, Jonathan Nakashima, Kristein King, Trivia Frazier. A patient derived xenograft model for lung adenocarcinoma using Obagel®, a human derived hydrogel. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4669.
U ntil recently, the complexity of adipose tissue and its physiological role was not well appreci... more U ntil recently, the complexity of adipose tissue and its physiological role was not well appreciated. This changed with the discovery of adipokines such as leptin. The cellular composition of adipose tissue is heterogeneous and changes as a function of diabetes and disease states such as diabetes. Tissue engineers view adipose tissue as a rich source of adult stromal/stem cells isolated by collagenase digestion. In vitro and in vivo studies have documented that adipose stromal/stem cells are multipotent, with the ability to differentiate along the adipocyte, chondrocyte, osteoblast and other lineage pathways. The adipose stromal/stem cells secrete a wide range of cytokines and growth factors with potential paracrine actions. Furthermore, adipose stromal/stem cells exert immunomodulatory functions when added to mixed lymphocyte reactions, suggesting that they can be transplanted allogeneically. This review article focuses on these mechanisms of adipose stromal/stem cell action and their potential utility as cellular therapeutics.
Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delive... more Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delivery of cells and drugs. Existing hydrogel classifications include synthetic, recombinant, chemically defined, plant‐ or animal‐based, and tissue‐derived matrices. There is a need for materials that can support both human tissue modeling and clinically relevant applications requiring stiffness tunability. Human‐derived hydrogels are not only clinically relevant, but they also minimize the use of animal models for pre‐clinical studies. This study aims to characterize XGel, a new human‐derived hydrogel as an alternative to current murine‐derived and synthetic recombinant hydrogels that features unique physiochemical, biochemical, and biological properties that support adipocyte and bone differentiation. Rheology studies determine the viscosity, stiffness, and gelation features of XGel. Quantitative studies for quality control support consistency in the protein content between lots. Proteomics studies reveal that XGel is predominantly composed of extracellular matrix proteins, including fibrillin, collagens I–VI, and fibronectin. Electron microscopy of the hydrogel provides phenotypic characteristics in terms of porosity and fiber size. The hydrogel demonstrates biocompatibility as a coating material and as a 3D scaffold for the growth of multiple cell types. The results provide insight into the biological compatibility of this human‐derived hydrogel for tissue engineering.
Background Bone defect causes softening of bone, reduction of bone density and mass, and degenera... more Background Bone defect causes softening of bone, reduction of bone density and mass, and degenerating bone microstructure which directly leads to bone fractures. Tissue-derived engineered cell-seeded hydrogel scaffolds are considered a promising strategy for the treatment of such defects. Among the most important objectives is to develop a unique biological tissue derived scaffolding material that is easily manipulated, efficient for cell attachment, and biocompatible, as well as supports cell growth, proliferation, and differentiation along osteogenic lineage. Methods This research describes a technique for the preparation of decellularized hydrogels. The detergent-based procedure using Triton-X-100 reported here effectively eliminates cellular proteins and antigens, as well as nucleic acid, while causing minimal damage to the extracellular matrix (ECM). The final product contains natural ECM components that guide stem cell differentiation by simulating the natural tissue microenvi...
Articular cartilage is composed of chondrocytes surrounded by a porous permeable extracellular ma... more Articular cartilage is composed of chondrocytes surrounded by a porous permeable extracellular matrix. It has a limited spontaneous healing capability post-injury which, if left untreated, can result in severe osteochondral disease. Currently, osteochondral (OC) defects are treated by bone marrow stimulation, artificial joint replacement, or transplantation of bone, cartilage, and periosteum, while autologous osteochondral transplantation is also an option; it carries the risk of donor site damage and is limited only to the treatment of small defects. Allografts may be used for larger defects; however, they have the potential to elicit an immune response. A possible alternative solution to treat osteochondral diseases involves the use of stromal/stem cells. Human adipose-derived stromal/stem cells (ASCs) can differentiate into cartilage and bone cells. The ASC can be combined with both natural and synthetic scaffolds to support cell delivery, growth, proliferation, migration, and di...
International regulatory agencies such as the Food and Drug Administration have mandated that the... more International regulatory agencies such as the Food and Drug Administration have mandated that the scientific community develop humanized microphysiological systems (MPS) as an in vitro alternative to animal models in the near future. While the breast cancer research community has long appreciated the importance of threedimensional growth dynamics in their experimental models, there are remaining obstacles preventing a full conversion to humanized MPS for drug discovery and pathophysiological studies. This perspective evaluates the current status of human tissue-derived cells and scaffolds as building blocks for an ''idealized'' breast cancer MPS based on bioengineering design principles. It considers the utility of adipose tissue as a potential source of endothelial, lymphohematopoietic, and stromal cells for the support of breast cancer epithelial cells. The relative merits of potential MPS scaffolds derived from adipose tissue, blood components, and synthetic biomaterials is evaluated relative to the current ''gold standard'' material, Matrigel, a murine chondrosarcomaderived basement membrane-enriched hydrogel. The advantages and limitations of a humanized breast cancer MPS are discussed in the context of in-process and destructive read-out assays.
Microphysiological systems (MPS) created with human-derived cells and biomaterial scaffolds offer... more Microphysiological systems (MPS) created with human-derived cells and biomaterial scaffolds offer a potential in vitro alternative to in vivo animal models. The adoption of three-dimensional MPS models has economic, ethical, regulatory, and scientific implications for the fields of regenerative medicine, metabolism/obesity, oncology, and pharmaceutical drug discovery. Key opinion leaders acknowledge that MPS tools are uniquely positioned to aid in the objective to reduce, refine, and eventually replace animal experimentation while improving the accuracy of the finding's clinical translation. Adipose tissue has proven to be an accessible and available source of human-derived stromal vascular fraction (SVF) cells, a heterogeneous population available at point of care, and adipose-derived stromal/stem cells, a relatively homogeneous population requiring plastic adherence and culture expansion of the SVF cells. The adipose-derived stromal/stem cells or SVF cells, in combination with human tissue or synthetic biomaterial scaffolds, can be maintained for extended culture periods as three-dimensional MPS models under angiogenic, stromal, adipogenic, or osteogenic conditions. This review highlights recent literature relating to the versatile use of adipose-derived cells as fundamental components of three-dimensional MPS models for discovery research and development. In this context, it compares the merits and limitations of the adipose-derived stromal/stem cells relative to SVF cell models and considers the likely directions that this emerging field of scientific discovery will take in the near future.
Monitoring wound progression over time is a critical aspect for studies focused on in-depth molec... more Monitoring wound progression over time is a critical aspect for studies focused on in-depth molecular analysis or on evaluating the efficacy of potential novel therapies. Histopathological analysis of wound biopsies can provide significant insight into healing dynamics, yet there is no standardized and reproducible scoring system currently available. The purpose of this study was to develop and statistically validate a scoring system based on parameters in each phase of healing that can be easily and accurately assessed using either Hematoxylin & Eosin (H&E) or Masson's Trichrome (MT) staining. These parameters included re-epithelization, epithelial thickness index, keratinization, granulation tissue thickness, remodeling, and the scar elevation index. The initial phase of the study was to (1) optimize and clarify healing parameters to limit investigator bias and variability; (2) compare the consistency of parameters assessed using H&E versus MT staining. During the validation phase of this study, the accuracy and reproducibility of this scoring system was independently iterated upon and validated in four different types of murine skin wound models (Excisional; punch biopsy; pressure ulcers; burn wounds). A total of n = 54 histology sections were randomized, blinded, and assigned to two groups of independent investigators (n = 5 per group) for analysis. The sensitivity of each parameter (ranging between 80% and 95%) is reported with illustrations on the appropriate assessment method using ImageJ software. In the validated scoring system, the lowest score (score:0) is associated with an open/unhealed wound as is evident immediately and within the first day postinjury, whereas the highest score (score:12) is associated with a completely closed and healed wound without excessive scarring. This study defines and describes the minimum recommended criteria for assessing wound healing dynamics using the SPOT skin wound score. The acronym SPOT refers to the academic and scientific institutions that were involved in the development of the scoring system, namely
Tissue engineering is a promising approach for the repair and regeneration of cartilaginous tissu... more Tissue engineering is a promising approach for the repair and regeneration of cartilaginous tissue. Appropriate three-dimensional scaffolding materials that mimic cartilage are ideal for the repair of chondral defects. The emerging decellularized tissue-based scaffolds have the potential to provide essential biochemical signals and structural integrity, which mimics the natural tissue environment and directs cellular fate. Umbilical cord-derived hydrogels function as 3D scaffolding material, which support adherence, proliferation, migration, and differentiation of cells due to their similar biochemical composition to cartilage. Therefore, the present study aimed to establish a protocol for the formulation of a hydrogel from decellularized human umbilical cord (DUC) tissue, and assess its application in the proliferation and differentiation of UC-MSCs along chondrogenic lineage. The results showed that the umbilical cord was efficiently decellularized. Subsequently, DUC hydrogel was ...
Human adipose-derived stromal/stem cells (hASC) are widely used for in vitro modeling of physiolo... more Human adipose-derived stromal/stem cells (hASC) are widely used for in vitro modeling of physiologically relevant human adipose tissue. These models are useful for the development of tissue constructs for soft tissue regeneration and 3-dimensional (3D) microphysiological systems (MPS) for drug discovery. In this systematic review, we report on the current state of hASC culture and assessment methods for adipose tissue engineering using 3D MPS. Our search efforts resulted in the identification of 184 independent records, of which 27 were determined to be most relevant to the goals of the present review. Our results demonstrate a lack of consensus on methods for hASC culture and assessment for the production of physiologically relevant in vitro models of human adipose tissue. Few studies have assessed the impact of different 3D culture conditions on hASC adipogenesis. Additionally, there has been a limited use of assays for characterizing the functionality of adipose tissue in vitro. ...
Human adipose-derived stem cells (ASCs) have potential to improve wound healing; however, their e... more Human adipose-derived stem cells (ASCs) have potential to improve wound healing; however, their equivalents from domestic animals have received less attention as an alternative cell-based therapy for animals or even humans. Hypoxia is essential for maintaining stem cell functionality in tissue-specific niches. However, a cellular response to low oxygen levels has not been demonstrated in pig ASCs. Hence, the goal of our study was to characterize ASCs isolated from the subcutaneous fat of domestic pigs (pASCs) and examine the effect of hypoxia on their proteome and functional characteristics that might reproduce pASCs wound healing ability. Analysis of immunophenotypic and functional markers demonstrated that pASCs exhibited characteristics of mesenchymal stem cells. Proteomic analysis revealed 70 differentially abundant proteins between pASCs cultured under hypoxia (1% O2) or normoxia (21% O2). Among them, 42 proteins were enriched in the cells exposed to low oxygen, whereas 28 prot...
Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial... more Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial challenge to healthcare delivery with particular impacts on geriatric, paraplegic, and quadriplegic demographics worldwide. Nevertheless, the current standard of care relies extensively on preventive measures to mitigate pressure injury, surgical debridement, skin flap procedures, and negative pressure wound vacuum measures. This article highlights the potential of adipose-, blood-, and cellulose-derived products (cells, decellularized matrices and scaffolds, and exosome and secretome factors) as a means to address this unmet medical need. The current status of this research area is evaluated and discussed in the context of promising avenues for future discovery.
Pressure ulcers (PUs) result in part due to ischemia-reperfusion injury to the skin and present f... more Pressure ulcers (PUs) result in part due to ischemia-reperfusion injury to the skin and present frequently in elderly or quadriplegic patients with reduced mobility. Despite the high economic and societal cost of this condition, PU therapy relies primarily on preventive strategies and invasive surgical intervention. A growing body of clinical literature suggests that localized injection of adipose-derived cells can accelerate and enhance the closure of PUs. The current study systematically evaluated the safety of human adipose stromal vascular fraction (SVF) cells isolated using a closed system device when injected into a murine PU injury model. The human SVF cells were characterized by colony-forming unit-fibroblast and differentiation assays before use. Young (2 months) immunocompetent C57BL/6 mice subjected to a magnet-induced ischemia-reperfusion injury were injected subcutaneously with human SVF cells at increasing doses (0.25-2 million cells). The size of the PU was monitored over a 20-day period. Both female and male mice tolerated the concentrationdependent injection of the SVF cells without complications. While male mice trended toward more rapid wound closure rates in response to lower SVF cell concentrations (0.25-0.5 million cells), female mice responded favorably to higher SVF cell concentrations (1-2 million cells); however, outcomes did not reach statistical significance in either sex. Overall, the study demonstrates that human SVF cells prepared with a closed system device designed for use at point of care can be safely administered for PU therapy in an immunocompetent host animal model.
Pressure injuries/ulcers are frequent complications in elderly, paraplegic, and quadriplegic pati... more Pressure injuries/ulcers are frequent complications in elderly, paraplegic, and quadriplegic patients that account for considerable cost to the international health care economy and remain refractory to current treatment options. Autologous or allogeneic adult stromal/stem cells represent an alternative therapeutic approach. The current study extends prior findings by exploring the safety and efficacy of human adipose-derived stromal/stem cells (ASC) therapy in an established immunocompetent murine skin pressure ulcer model where dermal fibroblast cells (DFC) served as a control. Human adipose tissue was processed using a closed system device designed for point-of-care use in the operating room and on file with the Food and Drug Administration. Cell characterization was performed using colony forming unit-fibroblast (CFU-F), differentiation, and immunophenotypic assays in vitro. Wound healing was assessed over a 20-day period based on photomicrographs, histology, and immunohistochemistry. The isolated human ASC displayed significantly greater colony formation relative to DFC while both populations exhibited comparable immunophenotype and differentiation potential. Both fresh and cryopreserved human ASC significantly accelerated and enhanced wound healing in young (2 month) mice of both sexes relative to DFC controls based on tissue architecture and CD68+ cell infiltration. In contrast, while injection of either fresh or cryopreserved human ASC was safe in older mice, the fresh ASC significantly enhanced wound closure relative to the cryopreserved ASC. Overall, these findings support the safety and efficacy of human ASC isolated using a closed-system device designed for clinical procedures in the future treatment of pressure injuries.
Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a... more Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a wide range of disease states; however, many expansion protocols rely on the use of fetal bovine serum (FBS) as a cell culture nutrient supplement. The current study explores the substitution of lysates from expired human platelets (HPLs) as an FBS substitute. Methods: Expired human platelets from an authorized blood center were lysed by freeze/thawing and used to examine human ASCs with respect to proliferation using hematocytometer cell counts, colony forming unit-fibroblast (CFU-F) frequency, surface immunophenotype by flow cytometry, and tri-lineage (adipocyte, chondrocyte, osteoblast) differentiation potential by histochemical staining. Results: The proliferation assays demonstrated that HPLs supported ASC proliferation in a concentration dependent manner, reaching levels that exceeded that observed in the presence of 10% FBS. The concentration of 0.75% HPLs was equivalent to 10% FB...
ABSTRACT The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the for... more ABSTRACT The delivery of curcumin, a broad-spectrum anticancer drug, has been explored in the form of liposomal nanoparticles to treat osteosarcoma (OS). Curcumin is water insoluble and an effective delivery route is through encapsulation in cyclodextrins followed by a second encapsulation in liposomes. Liposomal curcumin's potential was evaluated against cancer models of mesenchymal (OS) and epithelial origin (breast cancer). The resulting 2-Hydroxypropyl-γ-cyclodextrin/curcumin - liposome complex shows promising anticancer potential both in vitro and in vivo against KHOS OS cell line and MCF-7 breast cancer cell line. An interesting aspect is that liposomal curcumin initiates the caspase cascade that leads to apoptotic cell death in vitro in comparison with DMSO-curcumin induced autophagic cell death. In addition, the efficiency of the liposomal curcumin formulation was confirmed in vivo using a xenograft OS model. Curcumin-loaded γ-cyclodextrin liposomes indicate significant potential as delivery vehicles for the treatment of cancers of different tissue origin. FROM THE CLINICAL EDITOR: Curcumin-loaded γ-cyclodextrin liposomes were demonstrated in vitro to have significant potential as delivery vehicles for the treatment of cancers of mesenchymal and epithelial origin. Differences between mechanisms of cell death were also evaluated.
Patient Derived Xenografts (PDXs) are valuable disease models as they preserve the tumor microenv... more Patient Derived Xenografts (PDXs) are valuable disease models as they preserve the tumor microenvironment’s structure, providing the most clinically relevant in vivo testing platform. Nevertheless, most of those models still use animal derived hydrogels as scaffolds for patient derived cells, reducing the potential to fully mimic the physiological microenvironment of the disease and human condition. ObaGel® is a human-derived hydrogel successfully deployed for supporting 3D cell culture, organoid, perfusion bioreactor, and/microphysiological system development for studying obesity, type II diabetes, and various forms of related cancers. Here we are investigating the use of Obagel® in the in-vivo development of a PDX-derived culture of lung adenocarcinoma and compare its performance to currently used animal derived hydrogels. Methods: 3 × 10^6 CRT00295 Non-small cell lung adenocarcinoma cells in 100ul 50:50 indicated Gel in PBS per injection site; the study used 10 NOG mice per condition. Results: The results indicated that ObaGel® performed similarly in terms of engraftment and tumor kinetics compared to animal derived hydrogels such as Cultrex, Geltrex, and Matrigel, using a patient-derived xenograft culture of lung adenocarcinoma. Conclusions: Our data supports the use of a human-derived hydrogel for PDX models, ObaGel® performs equivalently to animal derived hydrogels to support tumor volume over time. The use of a human derived hydrogels as scaffolds for PDX models provide a better humanized local microenvironment for lung adenocarcinoma. Obagel® can also be adapted for use as xenografts in other tissue/disease models Citation Format: Cecilia Sanchez, Jonathan Nakashima, Kristein King, Trivia Frazier. A patient derived xenograft model for lung adenocarcinoma using Obagel®, a human derived hydrogel. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4669.
U ntil recently, the complexity of adipose tissue and its physiological role was not well appreci... more U ntil recently, the complexity of adipose tissue and its physiological role was not well appreciated. This changed with the discovery of adipokines such as leptin. The cellular composition of adipose tissue is heterogeneous and changes as a function of diabetes and disease states such as diabetes. Tissue engineers view adipose tissue as a rich source of adult stromal/stem cells isolated by collagenase digestion. In vitro and in vivo studies have documented that adipose stromal/stem cells are multipotent, with the ability to differentiate along the adipocyte, chondrocyte, osteoblast and other lineage pathways. The adipose stromal/stem cells secrete a wide range of cytokines and growth factors with potential paracrine actions. Furthermore, adipose stromal/stem cells exert immunomodulatory functions when added to mixed lymphocyte reactions, suggesting that they can be transplanted allogeneically. This review article focuses on these mechanisms of adipose stromal/stem cell action and their potential utility as cellular therapeutics.
Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delive... more Hydrogels are 3D scaffolds used as alternatives to in vivo models for disease modeling and delivery of cells and drugs. Existing hydrogel classifications include synthetic, recombinant, chemically defined, plant‐ or animal‐based, and tissue‐derived matrices. There is a need for materials that can support both human tissue modeling and clinically relevant applications requiring stiffness tunability. Human‐derived hydrogels are not only clinically relevant, but they also minimize the use of animal models for pre‐clinical studies. This study aims to characterize XGel, a new human‐derived hydrogel as an alternative to current murine‐derived and synthetic recombinant hydrogels that features unique physiochemical, biochemical, and biological properties that support adipocyte and bone differentiation. Rheology studies determine the viscosity, stiffness, and gelation features of XGel. Quantitative studies for quality control support consistency in the protein content between lots. Proteomics studies reveal that XGel is predominantly composed of extracellular matrix proteins, including fibrillin, collagens I–VI, and fibronectin. Electron microscopy of the hydrogel provides phenotypic characteristics in terms of porosity and fiber size. The hydrogel demonstrates biocompatibility as a coating material and as a 3D scaffold for the growth of multiple cell types. The results provide insight into the biological compatibility of this human‐derived hydrogel for tissue engineering.
Background Bone defect causes softening of bone, reduction of bone density and mass, and degenera... more Background Bone defect causes softening of bone, reduction of bone density and mass, and degenerating bone microstructure which directly leads to bone fractures. Tissue-derived engineered cell-seeded hydrogel scaffolds are considered a promising strategy for the treatment of such defects. Among the most important objectives is to develop a unique biological tissue derived scaffolding material that is easily manipulated, efficient for cell attachment, and biocompatible, as well as supports cell growth, proliferation, and differentiation along osteogenic lineage. Methods This research describes a technique for the preparation of decellularized hydrogels. The detergent-based procedure using Triton-X-100 reported here effectively eliminates cellular proteins and antigens, as well as nucleic acid, while causing minimal damage to the extracellular matrix (ECM). The final product contains natural ECM components that guide stem cell differentiation by simulating the natural tissue microenvi...
Articular cartilage is composed of chondrocytes surrounded by a porous permeable extracellular ma... more Articular cartilage is composed of chondrocytes surrounded by a porous permeable extracellular matrix. It has a limited spontaneous healing capability post-injury which, if left untreated, can result in severe osteochondral disease. Currently, osteochondral (OC) defects are treated by bone marrow stimulation, artificial joint replacement, or transplantation of bone, cartilage, and periosteum, while autologous osteochondral transplantation is also an option; it carries the risk of donor site damage and is limited only to the treatment of small defects. Allografts may be used for larger defects; however, they have the potential to elicit an immune response. A possible alternative solution to treat osteochondral diseases involves the use of stromal/stem cells. Human adipose-derived stromal/stem cells (ASCs) can differentiate into cartilage and bone cells. The ASC can be combined with both natural and synthetic scaffolds to support cell delivery, growth, proliferation, migration, and di...
International regulatory agencies such as the Food and Drug Administration have mandated that the... more International regulatory agencies such as the Food and Drug Administration have mandated that the scientific community develop humanized microphysiological systems (MPS) as an in vitro alternative to animal models in the near future. While the breast cancer research community has long appreciated the importance of threedimensional growth dynamics in their experimental models, there are remaining obstacles preventing a full conversion to humanized MPS for drug discovery and pathophysiological studies. This perspective evaluates the current status of human tissue-derived cells and scaffolds as building blocks for an ''idealized'' breast cancer MPS based on bioengineering design principles. It considers the utility of adipose tissue as a potential source of endothelial, lymphohematopoietic, and stromal cells for the support of breast cancer epithelial cells. The relative merits of potential MPS scaffolds derived from adipose tissue, blood components, and synthetic biomaterials is evaluated relative to the current ''gold standard'' material, Matrigel, a murine chondrosarcomaderived basement membrane-enriched hydrogel. The advantages and limitations of a humanized breast cancer MPS are discussed in the context of in-process and destructive read-out assays.
Microphysiological systems (MPS) created with human-derived cells and biomaterial scaffolds offer... more Microphysiological systems (MPS) created with human-derived cells and biomaterial scaffolds offer a potential in vitro alternative to in vivo animal models. The adoption of three-dimensional MPS models has economic, ethical, regulatory, and scientific implications for the fields of regenerative medicine, metabolism/obesity, oncology, and pharmaceutical drug discovery. Key opinion leaders acknowledge that MPS tools are uniquely positioned to aid in the objective to reduce, refine, and eventually replace animal experimentation while improving the accuracy of the finding's clinical translation. Adipose tissue has proven to be an accessible and available source of human-derived stromal vascular fraction (SVF) cells, a heterogeneous population available at point of care, and adipose-derived stromal/stem cells, a relatively homogeneous population requiring plastic adherence and culture expansion of the SVF cells. The adipose-derived stromal/stem cells or SVF cells, in combination with human tissue or synthetic biomaterial scaffolds, can be maintained for extended culture periods as three-dimensional MPS models under angiogenic, stromal, adipogenic, or osteogenic conditions. This review highlights recent literature relating to the versatile use of adipose-derived cells as fundamental components of three-dimensional MPS models for discovery research and development. In this context, it compares the merits and limitations of the adipose-derived stromal/stem cells relative to SVF cell models and considers the likely directions that this emerging field of scientific discovery will take in the near future.
Monitoring wound progression over time is a critical aspect for studies focused on in-depth molec... more Monitoring wound progression over time is a critical aspect for studies focused on in-depth molecular analysis or on evaluating the efficacy of potential novel therapies. Histopathological analysis of wound biopsies can provide significant insight into healing dynamics, yet there is no standardized and reproducible scoring system currently available. The purpose of this study was to develop and statistically validate a scoring system based on parameters in each phase of healing that can be easily and accurately assessed using either Hematoxylin & Eosin (H&E) or Masson's Trichrome (MT) staining. These parameters included re-epithelization, epithelial thickness index, keratinization, granulation tissue thickness, remodeling, and the scar elevation index. The initial phase of the study was to (1) optimize and clarify healing parameters to limit investigator bias and variability; (2) compare the consistency of parameters assessed using H&E versus MT staining. During the validation phase of this study, the accuracy and reproducibility of this scoring system was independently iterated upon and validated in four different types of murine skin wound models (Excisional; punch biopsy; pressure ulcers; burn wounds). A total of n = 54 histology sections were randomized, blinded, and assigned to two groups of independent investigators (n = 5 per group) for analysis. The sensitivity of each parameter (ranging between 80% and 95%) is reported with illustrations on the appropriate assessment method using ImageJ software. In the validated scoring system, the lowest score (score:0) is associated with an open/unhealed wound as is evident immediately and within the first day postinjury, whereas the highest score (score:12) is associated with a completely closed and healed wound without excessive scarring. This study defines and describes the minimum recommended criteria for assessing wound healing dynamics using the SPOT skin wound score. The acronym SPOT refers to the academic and scientific institutions that were involved in the development of the scoring system, namely
Tissue engineering is a promising approach for the repair and regeneration of cartilaginous tissu... more Tissue engineering is a promising approach for the repair and regeneration of cartilaginous tissue. Appropriate three-dimensional scaffolding materials that mimic cartilage are ideal for the repair of chondral defects. The emerging decellularized tissue-based scaffolds have the potential to provide essential biochemical signals and structural integrity, which mimics the natural tissue environment and directs cellular fate. Umbilical cord-derived hydrogels function as 3D scaffolding material, which support adherence, proliferation, migration, and differentiation of cells due to their similar biochemical composition to cartilage. Therefore, the present study aimed to establish a protocol for the formulation of a hydrogel from decellularized human umbilical cord (DUC) tissue, and assess its application in the proliferation and differentiation of UC-MSCs along chondrogenic lineage. The results showed that the umbilical cord was efficiently decellularized. Subsequently, DUC hydrogel was ...
Human adipose-derived stromal/stem cells (hASC) are widely used for in vitro modeling of physiolo... more Human adipose-derived stromal/stem cells (hASC) are widely used for in vitro modeling of physiologically relevant human adipose tissue. These models are useful for the development of tissue constructs for soft tissue regeneration and 3-dimensional (3D) microphysiological systems (MPS) for drug discovery. In this systematic review, we report on the current state of hASC culture and assessment methods for adipose tissue engineering using 3D MPS. Our search efforts resulted in the identification of 184 independent records, of which 27 were determined to be most relevant to the goals of the present review. Our results demonstrate a lack of consensus on methods for hASC culture and assessment for the production of physiologically relevant in vitro models of human adipose tissue. Few studies have assessed the impact of different 3D culture conditions on hASC adipogenesis. Additionally, there has been a limited use of assays for characterizing the functionality of adipose tissue in vitro. ...
Human adipose-derived stem cells (ASCs) have potential to improve wound healing; however, their e... more Human adipose-derived stem cells (ASCs) have potential to improve wound healing; however, their equivalents from domestic animals have received less attention as an alternative cell-based therapy for animals or even humans. Hypoxia is essential for maintaining stem cell functionality in tissue-specific niches. However, a cellular response to low oxygen levels has not been demonstrated in pig ASCs. Hence, the goal of our study was to characterize ASCs isolated from the subcutaneous fat of domestic pigs (pASCs) and examine the effect of hypoxia on their proteome and functional characteristics that might reproduce pASCs wound healing ability. Analysis of immunophenotypic and functional markers demonstrated that pASCs exhibited characteristics of mesenchymal stem cells. Proteomic analysis revealed 70 differentially abundant proteins between pASCs cultured under hypoxia (1% O2) or normoxia (21% O2). Among them, 42 proteins were enriched in the cells exposed to low oxygen, whereas 28 prot...
Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial... more Acute and chronic skin wounds due to burns, pressure injuries, and trauma represent a substantial challenge to healthcare delivery with particular impacts on geriatric, paraplegic, and quadriplegic demographics worldwide. Nevertheless, the current standard of care relies extensively on preventive measures to mitigate pressure injury, surgical debridement, skin flap procedures, and negative pressure wound vacuum measures. This article highlights the potential of adipose-, blood-, and cellulose-derived products (cells, decellularized matrices and scaffolds, and exosome and secretome factors) as a means to address this unmet medical need. The current status of this research area is evaluated and discussed in the context of promising avenues for future discovery.
Pressure ulcers (PUs) result in part due to ischemia-reperfusion injury to the skin and present f... more Pressure ulcers (PUs) result in part due to ischemia-reperfusion injury to the skin and present frequently in elderly or quadriplegic patients with reduced mobility. Despite the high economic and societal cost of this condition, PU therapy relies primarily on preventive strategies and invasive surgical intervention. A growing body of clinical literature suggests that localized injection of adipose-derived cells can accelerate and enhance the closure of PUs. The current study systematically evaluated the safety of human adipose stromal vascular fraction (SVF) cells isolated using a closed system device when injected into a murine PU injury model. The human SVF cells were characterized by colony-forming unit-fibroblast and differentiation assays before use. Young (2 months) immunocompetent C57BL/6 mice subjected to a magnet-induced ischemia-reperfusion injury were injected subcutaneously with human SVF cells at increasing doses (0.25-2 million cells). The size of the PU was monitored over a 20-day period. Both female and male mice tolerated the concentrationdependent injection of the SVF cells without complications. While male mice trended toward more rapid wound closure rates in response to lower SVF cell concentrations (0.25-0.5 million cells), female mice responded favorably to higher SVF cell concentrations (1-2 million cells); however, outcomes did not reach statistical significance in either sex. Overall, the study demonstrates that human SVF cells prepared with a closed system device designed for use at point of care can be safely administered for PU therapy in an immunocompetent host animal model.
Pressure injuries/ulcers are frequent complications in elderly, paraplegic, and quadriplegic pati... more Pressure injuries/ulcers are frequent complications in elderly, paraplegic, and quadriplegic patients that account for considerable cost to the international health care economy and remain refractory to current treatment options. Autologous or allogeneic adult stromal/stem cells represent an alternative therapeutic approach. The current study extends prior findings by exploring the safety and efficacy of human adipose-derived stromal/stem cells (ASC) therapy in an established immunocompetent murine skin pressure ulcer model where dermal fibroblast cells (DFC) served as a control. Human adipose tissue was processed using a closed system device designed for point-of-care use in the operating room and on file with the Food and Drug Administration. Cell characterization was performed using colony forming unit-fibroblast (CFU-F), differentiation, and immunophenotypic assays in vitro. Wound healing was assessed over a 20-day period based on photomicrographs, histology, and immunohistochemistry. The isolated human ASC displayed significantly greater colony formation relative to DFC while both populations exhibited comparable immunophenotype and differentiation potential. Both fresh and cryopreserved human ASC significantly accelerated and enhanced wound healing in young (2 month) mice of both sexes relative to DFC controls based on tissue architecture and CD68+ cell infiltration. In contrast, while injection of either fresh or cryopreserved human ASC was safe in older mice, the fresh ASC significantly enhanced wound closure relative to the cryopreserved ASC. Overall, these findings support the safety and efficacy of human ASC isolated using a closed-system device designed for clinical procedures in the future treatment of pressure injuries.
Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a... more Introduction: Adipose derived stromal/stem cells (ASCs) hold potential as cell therapeutics for a wide range of disease states; however, many expansion protocols rely on the use of fetal bovine serum (FBS) as a cell culture nutrient supplement. The current study explores the substitution of lysates from expired human platelets (HPLs) as an FBS substitute. Methods: Expired human platelets from an authorized blood center were lysed by freeze/thawing and used to examine human ASCs with respect to proliferation using hematocytometer cell counts, colony forming unit-fibroblast (CFU-F) frequency, surface immunophenotype by flow cytometry, and tri-lineage (adipocyte, chondrocyte, osteoblast) differentiation potential by histochemical staining. Results: The proliferation assays demonstrated that HPLs supported ASC proliferation in a concentration dependent manner, reaching levels that exceeded that observed in the presence of 10% FBS. The concentration of 0.75% HPLs was equivalent to 10% FB...
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Papers by Trivia Frazier