Biophysical Parameters Can Induce Epithelial-to-Mesenchymal Phenotypic and Genotypic Changes in HT-29 Cells: A Preliminary Study.

Epithelial to mesenchymal transition (EMT) in cancer is the process described where cancer epithelial cells acquire mesenchymal properties which can lead to enhanced invasiveness. Three-dimensional cancer models often lack the relevant and biomimetic microenvironment parameters appropriate to the native tumour microenvironment thought to drive EMT. In this study, HT-29 epithelial colorectal cells were cultivated in different oxygen and collagen concentrations to investigate how these biophysical parameters influenced invasion patterns and EMT. Colorectal HT-29 cells were grown in physiological hypoxia (5% O2) and normoxia (21% O2) in 2D, 3D soft (60 Pa), and 3D stiff (4 kPa) collagen matrices. Physiological hypoxia was sufficient to trigger expression of markers of EMT in the HT-29 cells in 2D by day 7. This is in contrast to a control breast cancer cell line, MDA-MB-231, which expresses a mesenchymal phenotype regardless of the oxygen concentration. In 3D, HT-29 cells invaded more extensively in a stiff matrix environment with corresponding increases in the invasive genes MMP2 and RAE1. This demonstrates that the physiological environment can directly impact HT-29 cells in terms of EMT marker expression and invasion, compared to an established cell line, MDA-MB-231, which has already undergone EMT. This study highlights the importance of the biophysical microenvironment to cancer epithelial cells and how these factors can direct cell behaviour. In particular, that stiffness of the 3D matrix drives greater invasion in HT-29 cells regardless of hypoxia. It is also pertinent that some cell lines (already having undergone EMT) are not as sensitive to the biophysical features of their microenvironment.

Evaluating Oxygen Tensions Related to Bone Marrow and Matrix for MSC Differentiation in 2D and 3D Biomimetic Lamellar Scaffolds.

The physiological O2 microenvironment of mesenchymal stem cells (MSCs) and osteoblasts and the dimensionality of a substrate are known to be important in regulating cell phenotype and function. By providing the physiologically normoxic environments of bone marrow (5%) and matrix (12%), we assessed their potential to maintain stemness, induce osteogenic differentiation, and enhance the material properties in the micropatterned collagen/silk fibroin scaffolds that were produced in 2D or 3D. Expression of osterix (OSX) and vascular endothelial growth factor A (VEGFA) was significantly enhanced in the 3D scaffold in all oxygen environments. At 21% O2, OSX and VEGFA expressions in the 3D scaffold were respectively 13,200 and 270 times higher than those of the 2D scaffold. Markers for assessing stemness were significantly more pronounced on tissue culture polystyrene and 2D scaffold incubated at 5% O2. At 21% O2, we measured significant increases in ultimate tensile strength (p < 0.0001) and Young's modulus (p = 0.003) of the 3D scaffold compared to the 2D scaffold, whilst 5% O2 hindered the positive effect of cell seeding on tensile strength. In conclusion, we demonstrated that the 3D culture of MSCs in collagen/silk fibroin scaffolds provided biomimetic cues for bone progenitor cells toward differentiation and enhanced the tensile mechanical properties.

Tissue-Engineering the Fibrous Pancreatic Tumour Stroma Capsule in 3D Tumouroids to Demonstrate Paclitaxel Response.

Pancreatic cancer is a unique cancer in that up to 90% of its tumour mass is composed of a hypovascular and fibrotic stroma. This makes it extremely difficult for chemotherapies to be delivered into the core of the cancer mass. We tissue-engineered a biomimetic 3D pancreatic cancer ("tumouroid") model comprised of a central artificial cancer mass (ACM), containing MIA Paca-2 cells, surrounded by a fibrotic stromal compartment. This stromal compartment had a higher concentration of collagen type I, fibronectin, laminin, and hyaluronic acid (HA) than the ACM. The incorporation of HA was validated with alcian blue staining. Response to paclitaxel was determined in 2D MIA Paca-2 cell cultures, the ACMs alone, and in simple and complex tumouroids, in order to demonstrate drug sensitivity within pancreatic tumouroids of increasing complexity. The results showed that MIA Paca-2 cells grew into the complex stroma and invaded as cell clusters with a maximum distance of 363.7 µm by day 21. In terms of drug response, the IC50 for paclitaxel for MIA Paca-2 cells increased from 0.819 nM in 2D to 3.02 nM in ACMs and to 5.87 nM and 3.803 nM in simple and complex tumouroids respectively, indicating that drug penetration may be significantly reduced in the latter. The results demonstrate the need for biomimetic models during initial drug testing and evaluation.

Cancer-associated fibroblasts mediate cancer progression and remodel the tumouroid stroma.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are highly differentiated and heterogeneous cancer-stromal cells that promote tumour growth, angiogenesis and matrix remodelling. METHODS: We utilised an adapted version of a previously developed 3D in vitro model of colorectal cancer, composed of a cancer mass and the surrounding stromal compartment. We compared cancer invasion with an acellular stromal surround, a "healthy" or normal cellular stroma and a cancerous stroma. For the cancerous stroma, we incorporated six patient-derived CAF samples to study their differential effects on cancer growth, vascular network formation and remodelling. RESULTS: CAFs enhanced the distance and surface area of the invasive cancer mass whilst inhibiting vascular-like network formation. These processes correlated with the upregulation of hepatocyte growth factor (HGF), metallopeptidase inhibitor 1 (TIMP1) and fibulin-5 (FBLN5). Vascular remodelling of previously formed endothelial structures occurred through the disruption of complex networks, and was associated with the upregulation of vascular endothelial growth factor (VEGFA) and downregulation in vascular endothelial cadherin (VE-Cadherin). CONCLUSIONS: These results support, within a biomimetic 3D, in vitro framework, the direct role of CAFs in promoting cancer invasion, and their key function in driving vasculogenesis and angiogenesis.

Mapping human serum-induced gene networks as a basis for the creation of biomimetic periosteum for bone repair.

BACKGROUND: The periosteum is a highly vascularized, collagen-rich tissue that plays a crucial role in directing bone repair. This is orchestrated primarily by its resident progenitor cell population. Indeed, preservation of periosteum integrity is critical for bone healing. Cells extracted from the periosteum retain their osteochondrogenic properties and as such are a promising basis for tissue engineering strategies for the repair of bone defects. However, the culture expansion conditions and the way in which the cells are reintroduced to the defect site are critical aspects of successful translation. Indeed, expansion in human serum and implantation on biomimetic materials has previously been shown to improve in vivo bone formation. AIM: This study aimed to develop a protocol to allow for the expansion of human periosteum derived cells (hPDCs) in a biomimetic periosteal-like environment. METHODS: The expansion conditions were defined through the investigation of the bioactive cues involved in augmenting hPDC proliferative and multipotency characteristics, based on transcriptomic analysis of cells cultured in human serum. RESULTS: Master regulators of transcriptional networks were identified, and an optimized periosteum-derived growth factor cocktail (PD-GFC; containing ß-estradiol, FGF2, TNFα, TGFß, IGF-1 and PDGF-BB) was generated. Expansion of hPDCs in PD-GFC resulted in serum mimicry with regard to the cell morphology, proliferative capacity and chondrogenic differentiation. When incorporated into a three-dimensional collagen type 1 matrix and cultured in PD-GFC, the hPDCs migrated to the surface that represented the matrix topography of the periosteum cambium layer. Furthermore, gene expression analysis revealed a down-regulated WNT and TGFß signature and an up-regulation of CREB, which may indicate the hPDCs are recreating their progenitor cell signature. CONCLUSION: This study highlights the first stage in the development of a biomimetic periosteum, which may have applications in bone repair.

Initial validation of equilibrium contrast imaging for extracellular volume quantification using a three-dimensional engineered tissue model.

BACKGROUND: To test the principles underpinning equilibrium contrast imaging estimation of tissue extracellular volume (ECV) fraction, using a three-dimensional (3D) engineered tissue model with known cellular and extracellular volumes. METHODS: Six 3D tissue models (tumoroids) consisting of cell cultures within a collagen containing hydrogel were constructed after culture centrifugation and direct measurement of the cell component volume. Measured tumoroid ECV ranged from 0.89 to 1. ECV was calculated after measuring the T1 relaxation time at 3 Tesla using inversion recovery relaxometry (TI 100-1500 ms) within the tumoroids and surrounding medium before and 375 min after spiking the medium with Gadolinium (to achieve a concentration of 1.4 mM/L). Linear regression model prediction of directly measured ECV (ECVm ) by EQ-MRI measured ECV (ECVeq ); and Bland-Altman agreement between measures was assessed. RESULTS: The fractional cellular volume measured by EQ-MRI (ECVeq ) within the tumoroids ranged from 0.821 to 0.963. ECVeq was a good predictor of ECVm (R2 = 0.77, P = 0.02). The regression line Y-axis intercept (when X = 0) was 0.045 ± 0.019 with a slope of 1.28 ± 0.35. Bland-Altman comparison demonstrated 95% limits of agreement between -0.002 and 0.114 with a bias (SD) of 0.056 (0.03). CONCLUSION: This study supports the principles of ECV estimation using equilibrium contrast MRI, but future development of this model may allow validation over a wider, more physiological ECV range and a greater understanding of the effect of tissue extracellular protein burden on ECV.

Laminin promotes vascular network formation in 3D in vitro collagen scaffolds by regulating VEGF uptake.

Angiogenesis is an essential neovascularisation process, which if recapitulated in 3D in vitro, will provide better understanding of endothelial cell (EC) behaviour. Various cell types and growth factors are involved, with vascular endothelial growth factor (VEGF) and its receptors VEGFR1 and VEGFR2 key components. We were able to control the aggregation pattern of ECs in 3D collagen hydrogels, by varying the matrix composition and/or having a source of cells signalling angiogenic proteins. These aggregation patterns reflect the different developmental pathways that ECs take to form different sized tubular structures. Cultures with added laminin and thus increased expression of α6 integrin showed a significant increase (p<0.05) in VEGFR2 positive ECs and increased VEGF uptake. This resulted in the end-to-end network aggregation of ECs. In cultures without laminin and therefore low α6 integrin expression, VEGFR2 levels and VEGF uptake were significantly lower (p<0.05). These ECs formed contiguous sheets, analogous to the 'wrapping' pathway in development. We have identified a key linkage between integrin expression on ECs and their uptake of VEGF, regulated by VEGFR2, resulting in different aggregation patterns in 3D.

A 3D in vitro cancer model as a platform for nanoparticle uptake and imaging investigations.

In order to maximize the potential of nanoparticles (NPs) in cancer imaging and therapy, their mechanisms of interaction with host tissue need to be fully understood. NP uptake is known to be dramatically influenced by the tumor microenvironment, and an imaging platform that could replicate in vivo cellular conditions would make big strides in NP uptake studies. Here, a novel NP uptake platform consisting of a tissue-engineered 3D in vitro cancer model (tumoroid), which mimics the microarchitecture of a solid cancer mass and stroma, is presented. As the tumoroid exhibits fundamental characteristics of solid cancer tissue and its cellular and biochemical parameters are controllable, it provides a real alternative to animal models. Furthermore, an X-ray fluorescence imaging system is developed to demonstrate 3D imaging of GNPs and to determine uptake efficiency within the tumoroid. This platform has implications for optimizing the targeted delivery of NPs to cells to benefit cancer diagnostics and therapy.

Effects of photochemical riboflavin-mediated crosslinks on the physical properties of collagen constructs and fibrils.

The use of collagen scaffold in tissue engineering is on the rise, as modifications to mechanical properties are becoming more effective in strengthening constructs whilst preserving the natural biocompatibility. The combined technique of plastic compression and cross-linking is known to increase the mechanical strength of the collagen construct. Here, a modified protocol for engineering these collagen constructs is used to bring together a plastic compression method, combined with controlled photochemical crosslinking using riboflavin as a photoinitiator. In order to ascertain the effects of the photochemical crosslinking approach and the impact of the crosslinks created upon the properties of the engineered collagen constructs, the constructs were characterized both at the macroscale and at the fibrillar level. The resulting constructs were found to have a 2.5 fold increase in their Young's modulus, reaching a value of 650 ± 73 kPa when compared to non-crosslinked control collagen constructs. This value is not yet comparable to that of native tendon, but it proves that combining a crosslinking methodology to collagen tissue engineering may offer a new approach to create stronger, biomimetic constructs. A notable outcome of crosslinking collagen with riboflavin is the collagen's greater affinity for water; it was demonstrated that riboflavin crosslinked collagen retains water for a longer period of time compared to non-cross-linked control samples. The affinity of the cross-linked collagen to water also resulted in an increase of individual collagen fibrils' cross-sectional area as function of the crosslinking. These changes in water affinity and fibril morphology induced by the process of crosslinking could indicate that the crosslinked chains created during the photochemical crosslinking process may act as intermolecular hydrophilic nanosprings. These intermolecular nanosprings would be responsible for a change in the fibril morphology to accommodate variable volume of water within the fibril.

Patient-specific colorectal-cancer-associated fibroblasts modulate tumor microenvironment mechanics.

Cancer-associated fibroblasts (CAFs) play a major role in reorganizing the physical tumor micro-environment and changing tissue stiffness. Herein, using an engineered three-dimensional (3D) model that mimics the tumor's native biomechanical environment, we characterized the changes in matrix stiffness caused by six patient-specific colorectal CAF populations. After 21 days of culture, atomic force microscopy (AFM) was performed to precisely measure the local changes in tissue stiffness. Each CAF population exhibited heterogeneity in remodeling capabilities, with some patient-derived cells stiffening the matrix and others softening it. Tissue stiffening was mainly attributed to active contraction of the matrix by the cells, whereas the softening was due to enzymatic activity of matrix-cleaving proteins. This measured heterogeneity was lost when the CAFs were cocultured with colorectal cancer cells, as all samples significantly soften the tissue. The interplay between cancer cells and CAFs was critical as it altered any heterogeneity exhibited by CAFs alone.

Spatial transcriptomic interrogation of the tumour-stroma boundary in a 3D engineered model of ameloblastoma.

Stromal cells are key components of the tumour microenvironment (TME) and their incorporation into 3D engineered tumour-stroma models is essential for tumour mimicry. By engineering tumouroids with distinct tumour and stromal compartments, it has been possible to identify how gene expression of tumour cells is altered and influenced by the presence of different stromal cells. Ameloblastoma is a benign epithelial tumour of the jawbone. In engineered, multi-compartment tumouroids spatial transcriptomics revealed an upregulation of oncogenes in the ameloblastoma transcriptome where osteoblasts were present in the stromal compartment (bone stroma). Where a gingival fibroblast stroma was engineered, the ameloblastoma tumour transcriptome revealed increased matrix remodelling genes. This study provides evidence to show the stromal-specific effect on tumour behaviour and illustrates the importance of engineering biologically relevant stroma for engineered tumour models. Our novel results show that an engineered fibroblast stroma causes the upregulation of matrix remodelling genes in ameloblastoma which directly correlates to measured invasion in the model. In contrast the presence of a bone stroma increases the expression of oncogenes by ameloblastoma cells.

Position Paper Progress in the development of biomimetic engineered human tissues.

Tissue engineering (TE) is the multi-disciplinary approach to building 3D human tissue equivalents in the laboratory. The advancement of medical sciences and allied scientific disciplines have aspired to engineer human tissues for three decades. To date there is limited use of TE tissues/organs as replacement body parts in humans. This position paper outlines advances in engineering of specific tissues and organs with tissue-specific challenges. This paper outlines the technologies most successful for engineering tissues and key areas of advancement.

3D In Vitro Models for Investigating the Role of Stiffness in Cancer Invasion.

BACKGROUND: Tumorigenesis is attributed to the interactions of cancer cells with the tumor microenvironment through both biochemical cues and physical stimuli. Increased matrix deposition and realignment of the collagen fibers are detected by cancer cells, inducing epithelial-to-mesenchymal transition, which in turn stimulates cell motility and invasiveness. METHODS: This review provides an overview of current research on the role of the physical microenvironment in cancer invasion. This was achieved by using a systematic approach and providing meta-analyses. Particular focus was placed on in vitro three-dimensional models of epithelial cancers. We investigated questions such as the effect of matrix stiffening, activation of stromal cells, and identified potential advances in mechano-based therapies. RESULTS: Meta-analysis revealed that 64% of studies report cancer invasion promotion as stiffness increases, while 36% report the opposite. Experimental approaches and data interpretations were varied, each affecting the invasion of cancer differently. Examples are the experimental timeframes used (24 h to 21 days), the type of polymer used (24 types), and choice of cell line (33 cell lines). The stiffness of the 3D matrices varied from 0.5 to 300 kPa and 19% of these matrices' stiffness were outside commonly accepted physiological range. 100% of the studies outside biological stiffness range (above 20 kPa) report that stiffness does not promote cancer invasion. CONCLUSIONS: Taking this analysis into account, we inform on the type of experimental approaches that could be the most relevant and provide what would be a standardized protocol and reporting strategy.

Enhanced Biomimetics of Three-Dimensional Osteosarcoma Models: A Scoping Review.

This scoping review evaluated 3D osteosarcoma (OS) models' biomimicry, examining their ability to mimic the tumour microenvironment (TME) and their drug sensitivity. Adhering to PRISMA-ScR guidelines, the systematic search revealed 293 studies, with 70 selected for final analysis. Overall, 64% of 3D OS models were scaffold-based, compared to self-generated spheroid models. Scaffolds generated using native matrix were most common (42%) with collagen I/hydroxyapatite predominating. Both scaffold-based and scaffold-free models were used equally for drug screening. The sensitivity of cancer cells in 3D was reported to be lower than that of cells in 2D in ~90% of the drug screening studies. This correlates with the observed upregulation of drug resistance. OS cells cultured in extracellular matrix (ECM)-mimetic scaffolds and native biomaterials were more resistant than cells in 2D. Co-cultures of OS and stromal cells in 3D models enhanced osteogenic differentiation, ECM remodelling, mineralisation, and angiogenesis, suggesting that tumour-stroma crosstalk promotes disease progression. Seven studies demonstrated selective toxicity of chemotherapeutics towards OS cells while sparing stromal cells, providing useful evidence for developing biomimetic tumour-stroma models to test selective drug toxicity. In conclusion, this review highlights the need to enhance biomimicry in 3D OS models for TME recapitulation, especially in testing novel therapeutics. Future research should explore innovative 3D biomimetic models, biomaterials, and advancements in personalised medicine.

Innovations in Stem Cell Therapy for Diabetic Wound Healing.

Significance: The global burden of diabetic wounds, particularly diabetic foot ulcers, continues to have large economic and social impact throughout the world. Current strategies are not sufficient to overcome this burden of disease. Finding newer, more advanced regenerative cell and tissue-based strategies to reduce morbidity remains paramount. Recent Advances: Recent advances in stem cell therapies are discussed. We also highlight the practical issues of translating these advancing technologies into the clinical setting. Critical Issues: We discuss the use of somatic and induced pluripotent stem cells and the stromal vascular fraction, as well as innovations, including the use of 3D bioprinting of skin. We also explore related issues of using regenerative techniques in clinical practice, including the current regulatory landscape and translatability of in vivo research. Future Directions: Advances in stem cell manipulation showcase the best therapeutic resources available to enhance mechanisms of wound healing such as angiogenesis, cell proliferation, and collagen synthesis; potential methods include changing the scaffold microenvironment, including relative oxygen tension, and the use of gene modification and nanotechnology. Secretome engineering, particularly the use of extracellular vesicles, may be another potential cell-derived therapeutic that may enable use of cell-free translational therapy.

Evaluating the Impact of a Biomimetic Mechanical Environment on Cancer Invasion and Matrix Remodeling.

The stiffness of tumors and their host tissues is much higher than most hydrogels, which are conventionally used to study in vitro cancer progression. The tumoroid assay is an engineered 3D in vitro tumor model that allows investigation of cancer cell invasion in an environment that is biomimetic in terms of extracellular matrix (ECM) composition and stiffness. Using this model, the change in matrix stiffness by epithelial colorectal cancer cells is systematically characterized by atomic force microscopy indentation tests. Less invasive epithelial cancer cells stiffen the tumor microenvironment while highly aggressive epithelial cancer cells show significant softening of the tumor microenvironment. Changes in stiffness are attributed to both cell-generated active forces as well as ECM degradation and remodeling. The degradation is in part attributed to the enzymatic activity of matrix metalloproteinases (MMPs) as demonstrated by the significant expression of MMP-2 and MMP-9 at both gene and protein levels. Targeting MMP activity through broad-spectrum drug inhibition (BB-94) reverses the changes in stiffness and also decreases cancer cell invasion. These results promote the idea of using mechano-based cancer therapies such as MMP inhibition.

Urokinase-type plasminogen activator (uPA) regulates invasion and matrix remodelling in colorectal cancer.

Background: Cancer cells remodel their local physical environment through processes of matrix reorganisation, deposition, stiffening and degradation. Urokinase-type plasminogen activator (uPA), which is encoded by the PLAU gene, is an extracellular proteolytic enzyme known to be involved in cancer progression and tumour microenvironment (TME) remodelling. Perturbing uPA therefore has a strong potential as a mechano-based cancer therapy. This work is a bioengineering investigation to validate whether 1) uPA is involved in matrix degradation and 2) preventing matrix degradation by targeting uPA can reduce cancer cell invasion and metastasis. Methods: To this aim, we used an engineered 3D in vitro model, termed the tumouroid, that appropriately mimics the tumour's native biophysical environment (3 kPa). A CRISPR-Cas9 mediated uPA knockout was performed to introduce a loss of function mutation in the gene coding sequence. Subsequently, to validate the translational potential of blocking uPA action, we tested a pharmacological inhibitor, UK-371,801. The changes in matrix stiffness were measured by atomic force microscopy (AFM). Invasion was quantified using images of the tumouroid, obtained after 21 days of culture. Results: We showed that uPA is highly expressed in invasive breast and colorectal cancers, and these invasive cancer cells locally degrade their TME. PLAU (uPA) gene knock-out (KO) completely stopped matrix remodelling and significantly reduced cancer invasion. Many invasive cancer gene markers were also downregulated in the PLAU KO tumouroids. Pharmacological inhibition of uPA showed similarly promising results, where matrix degradation was reduced and so was the cancer invasion. Conclusion: This work supports the role of uPA in matrix degradation. It demonstrates that the invasion of cancer cells was significantly reduced when enzymatic breakdown of the TME matrix was prevented. Collectively, this provides strong evidence of the effectiveness of targeting uPA as a mechano-based cancer therapy.

Stromal cells regulate mechanics of tumour spheroid.

The remarkable contractility and force generation ability exhibited by cancer cells empower them to overcome the resistance and steric hindrance presented by a three-dimensional, interconnected matrix. Cancer cells disseminate by actively remodelling and deforming their extracellular matrix (ECM). The process of tumour growth and its ECM remodelling have been extensively studied, but the effect of the cellular tumour microenvironment (TME) has been ignored in most studies that investigated tumour-cell-mediated ECM deformations and realignment. This study reports the integration of stromal cells in spheroid contractility assays that impacts the ECM remodelling and invasion abilities of cancer spheroids. To investigate this, we developed a novel multilayer in vitro assay that incorporates stromal cells and quantifies the contractile deformations that tumour spheroids exert on the ECM. We observed a negative correlation between the spheroid invasion potential and the levels of collagen deformation. The presence of stromal cells significantly increased cancer cell invasiveness and altered the cancer cells' ability to deform and realign collagen gel, due to upregulation of proinflammatory cytokines. Interestingly, this was observed consistently in both metastatic and non-metastatic cancer cells. Our findings contribute to a better understanding of the vital role played by the cellular TME in regulating the invasive outgrowth of cancer cells and underscore the potential of utilising matrix deformation measurements as a biophysical marker for evaluating invasiveness and informing targeted therapeutic opportunities.

Associated changes in stiffness of collagen scaffolds during osteoblast mineralisation and bone formation.

OBJECTIVE: Engineering bone in 3D is important for both regenerative medicine purposes and for the development of accurate in vitro models of bone tissue. The changing material stiffness of bone tissue had not yet been monitored throughout the process of mineralisation and bone nodule formation by osteoblasts either during in vitro engineering or in development perspective. RESULTS: Within this short research note, stiffness changes (Young's modulus) during in vitro bone formation by primary osteoblasts in dense collagen scaffolds were monitored using atomic force microscopy. Data analysis revealed significant stiffening of 3D bone cultures at day 5 and 8 that was correlated with the onset of mineral deposition (p < 0.00005).

Reprogramming bone progenitor identity and potency through control of collagen density and oxygen tension.

The biophysical microenvironment of the cell is being increasingly used to control cell signaling and to direct cell function. Herein, engineered 3D tuneable biomimetic scaffolds are used to control the cell microenvironment of Adipose-derived Mesenchymal Stromal Cells (AMSC), which exhibit a collagen density-specific profile for early and late stage bone cell lineage status. Cell potency was enhanced when AMSCs were cultured within low collagen density environments in hypoxic conditions. A transitional culture containing varied collagen densities in hypoxic conditions directed differential cell fate responses. The early skeletal progenitor identity (PDPN+CD146-CD73+CD164+) was rescued in the cells which migrated into low collagen density gels, with cells continuously exposed to the high collagen density gels displaying a transitioned bone-cartilage-stromal phenotype (PDPN+CD146+CD73-CD164-). This study uncovers the significant contributions of the physical and physiological cell environment and highlights a chemically independent methodology for reprogramming and isolating skeletal progenitor cells from an adipose-derived cell population.