Mobilizing Endogenous Progenitor Cells Using pSDF1α-Activated Scaffolds Accelerates Angiogenesis and Bone Repair in Critical-Sized Bone Defects.

Mobilizing endogenous progenitor cells to repair damaged tissue in situ has the potential to revolutionize the field of regenerative medicine, while the early establishment of a vascular network will ensure survival of newly generated tissue. In this study, a gene-activated scaffold containing a stromal derived factor 1α plasmid (pSDF1α), a pro-angiogenic gene that is also thought to be involved in the recruitment of mesenchymal stromal cells (MSCs) to sites of injury is described. It is shown that over-expression of SDF1α protein enhanced MSC recruitment and induced vessel-like structure formation by endothelial cells in vitro. When implanted subcutaneously, transcriptomic analysis reveals that endogenous MSCs are recruited and significant angiogenesis is stimulated. Just 1-week after implantation into a calvarial critical-sized bone defect, pSDF1α-activated scaffolds are recruited MSCs and rapidly activate angiogenic and osteogenic programs, upregulating Runx2, Dlx5, and Sp7. At the same time-point, pVEGF-activated scaffolds are recruited a variety of cell types, activating endochondral ossification. The early response induced by both scaffolds leads to complete bridging of the critical-sized bone defects within 4-weeks. The versatile cell-free gene-activated scaffold described in this study is capable of harnessing and enhancing the body's own regenerative capacity and has immense potential in a myriad of applications.

Dual Glyoxalase-1 and ß-Klotho Gene-Activated Scaffold Reduces Methylglyoxal and Reprograms Diabetic Adipose-Derived Stem Cells: Prospects in Improved Wound Healing.

Tissue engineering approaches aim to provide biocompatible scaffold supports that allow healing to progress often in healthy tissue. In diabetic foot ulcers (DFUs), hyperglycemia impedes ulcer regeneration, due to complications involving accumulations of cellular methylglyoxal (MG), a key component of oxidated stress and premature cellular aging which further limits repair. In this study, we aim to reduce MG using a collagen-chondroitin sulfate gene-activated scaffold (GAS) containing the glyoxalase-1 gene (GLO-1) to scavenge MG and anti-fibrotic ß-klotho to restore stem cell activity in diabetic adipose-derived stem cells (dADSCs). dADSCs were cultured on dual GAS constructs for 21 days in high-glucose media in vitro. Our results show that dADSCs cultured on dual GAS significantly reduced MG accumulation (-84%; p < 0.05) compared to the gene-free controls. Similar reductions in profibrotic proteins α-smooth muscle actin (-65%) and fibronectin (-76%; p < 0.05) were identified in dual GAS groups. Similar findings were observed in the expression of pro-scarring structural proteins collagen I (-62%), collagen IV (-70%) and collagen VII (-86%). A non-significant decrease in the expression of basement membrane protein E-cadherin (-59%) was noted; however, the dual GAS showed a significant increase in the expression of laminin (+300%). We conclude that dual GAS-containing Glo-1 and ß-klotho had a synergistic MG detoxification and anti-fibrotic role in dADSC's. This may be beneficial to provide better wound healing in DFUs by controlling the diabetic environment and rejuvenating the diabetic stem cells towards improved wound healing.

Dual delivery gene-activated scaffold directs fibroblast activity and keratinocyte epithelization.

Fibroblasts are the most abundant cell type in dermal skin and keratinocytes are the most abundant cell type in the epidermis; both play a crucial role in wound remodeling and maturation. We aim to assess the functionality of a novel dual gene activated scaffold (GAS) on human adult dermal fibroblasts (hDFs) and see how the secretome produced could affect human dermal microvascular endothelial cells (HDMVECs) and human epidermal keratinocyte (hEKs) growth and epithelization. Our GAS is a collagen chondroitin sulfate scaffold loaded with pro-angiogenic stromal derived factor (SDF-1α) and/or an anti-aging ß-Klotho plasmids. hDFs were grown on GAS for two weeks and compared to gene-free scaffolds. GAS produced a significantly better healing outcome in the fibroblasts than in the gene-free scaffold group. Among the GAS groups, the dual GAS induced the most potent pro-regenerative maturation in fibroblasts with a downregulation in proliferation (twofold, p < 0.05), fibrotic remodeling regulators TGF-ß1 (1.43-fold, p < 0.01) and CTGF (1.4-fold, p < 0.05), fibrotic cellular protein α-SMA (twofold, p < 0.05), and fibronectin matrix deposition (twofold, p < 0.05). The dual GAS secretome also showed enhancements of paracrine keratinocyte pro-epithelializing ability (1.3-fold, p < 0.05); basement membrane regeneration through laminin (6.4-fold, p < 0.005) and collagen IV (8.7-fold, p < 0.005) deposition. Our findings demonstrate enhanced responses in dual GAS containing hDFs by proangiogenic SDF-1α and ß-Klotho anti-fibrotic rejuvenating activities. This was demonstrated by activating hDFs on dual GAS to become anti-fibrotic in nature while eliciting wound repair basement membrane proteins; enhancing a proangiogenic HDMVECs paracrine signaling and greater epithelisation of hEKs.

Anti-Aging ß-Klotho Gene-Activated Scaffold Promotes Rejuvenative Wound Healing Response in Human Adipose-Derived Stem Cells.

Wound healing requires a tight orchestration of complex cellular events. Disruption in the cell-signaling events can severely impair healing. The application of biomaterial scaffolds has shown healing potential; however, the potential is insufficient for optimal wound maturation. This study explored the functional impact of a collagen-chondroitin sulfate scaffold functionalized with nanoparticles carrying an anti-aging gene ß-Klotho on human adipose-derived stem cells (ADSCs) for rejuvenative healing applications. We studied the response in the ADSCs in three phases: (1) transcriptional activities of pluripotency factors (Oct-4, Nanog and Sox-2), proliferation marker (Ki-67), wound healing regulators (TGF-ß3 and TGF-ß1); (2) paracrine bioactivity of the secretome generated by the ADSCs; and (3) regeneration of basement membrane (fibronectin, laminin, and collagen IV proteins) and expression of scar-associated proteins (α-SMA and elastin proteins) towards maturation. Overall, we found that the ß-Klotho gene-activated scaffold offers controlled activation of ADSCs' regenerative abilities. On day 3, the ADSCs on the gene-activated scaffold showed enhanced (2.5-fold) activation of transcription factor Oct-4 that was regulated transiently. This response was accompanied by a 3.6-fold increase in the expression of the anti-fibrotic gene TGF-ß3. Through paracrine signaling, the ADSCs-laden gene-activated scaffold also controlled human endothelial angiogenesis and pro-fibrotic response in dermal fibroblasts. Towards maturation, the ADSCs-laden gene-activated scaffold further showed an enhanced regeneration of the basement membrane through increases in laminin (2.1-fold) and collagen IV (8.8-fold) deposition. The ADSCs also expressed 2-fold lower amounts of the scar-associated α-SMA protein with improved qualitative elastin matrix deposition. Collectively, we determined that the ß-Klotho gene-activated scaffold possesses tremendous potential for wound healing and could advance stem cell-based therapy for rejuvenative healing applications.

SDF-1α gene-activated collagen scaffold enhances provasculogenic response in a coculture of human endothelial cells with human adipose-derived stromal cells.

Novel biomaterials can be used to provide a better environment for cross talk between vessel forming endothelial cells and wound healing instructor stem cells for tissue regeneration. This study seeks to investigate if a collagen scaffold containing a proangiogenic gene encoding for the chemokine stromal-derived factor-1 alpha (SDF-1α GAS) could be used to enhance functional responses in a coculture of human umbilical vein endothelial cells (HUVECs) and human adipose-derived stem/stromal cells (ADSCs). Functional responses were determined by (1) monitoring the amount of junctional adhesion molecule VE-cadherin released during 14 days culture, (2) expression of provasculogenic genes on the 14th day, and (3) the bioactivity of secreted factors on neurogenic human Schwann cells. When we compared our SDF-1α GAS with a gene-free scaffold, the results showed positive proangiogenic determination characterized by a transient yet controlled release of the VE-cadherin. On the 14th day, the coculture on the SDF-1α GAS showed enhanced maturation than its gene-free equivalent through the elevation of provasculogenic genes (SDF-1α-7.4-fold, CXCR4-1.5-fold, eNOS-1.5-fold). Furthermore, we also found that the coculture on SDF-1α GAS secretes bioactive factors that significantly (p < 0.01) enhanced human Schwann cells' clustering to develop toward Bünger band-like structures. Conclusively, this study reports that SDF-1α GAS could be used to produce a bioactive vascularized construct through the enhancement of the cooperative effects between endothelial cells and ADSCs.

SDF-1α Gene-Activated Collagen Scaffold Restores Pro-Angiogenic Wound Healing Features in Human Diabetic Adipose-Derived Stem Cells.

Non-healing diabetic foot ulcers (DFUs) can lead to leg amputation in diabetic patients. Autologous stem cell therapy holds some potential to solve this problem; however, diabetic stem cells are relatively dysfunctional and restrictive in their wound healing abilities. This study sought to explore if a novel collagen-chondroitin sulfate (coll-CS) scaffold, functionalized with polyplex nanoparticles carrying the gene encoding for stromal-derived factor-1 alpha (SDF-1α gene-activated scaffold), can enhance the regenerative functionality of human diabetic adipose-derived stem cells (ADSCs). We assessed the impact of the gene-activated scaffold on diabetic ADSCs by comparing their response against healthy ADSCs cultured on a gene-free scaffold over two weeks. Overall, we found that the gene-activated scaffold could restore the pro-angiogenic regenerative response in the human diabetic ADSCs similar to the healthy ADSCs on the gene-free scaffold. Gene and protein expression analysis revealed that the gene-activated scaffold induced the overexpression of SDF-1α in diabetic ADSCs and engaged the receptor CXCR7, causing downstream ß-arrestin signaling, as effectively as the transfected healthy ADSCs. The transfected diabetic ADSCs also exhibited pro-wound healing features characterized by active matrix remodeling of the provisional fibronectin matrix and basement membrane protein collagen IV. The gene-activated scaffold also induced a controlled pro-healing response in the healthy ADSCs by disabling early developmental factors signaling while promoting the expression of tissue remodeling components. Conclusively, we show that the SDF-1α gene-activated scaffold can overcome the deficiencies associated with diabetic ADSCs, paving the way for autologous stem cell therapies combined with novel biomaterials to treat DFUs.

SDF-1α gene-activated collagen scaffold drives functional differentiation of human Schwann cells for wound healing applications.

Enhancing angiogenesis is the prime target of current biomaterial-based wound healing strategies. However, these approaches largely overlook the angiogenic role of the cells of the nervous system. Therefore, we explored the role of a collagen-chondroitin sulfate scaffold functionalized with a proangiogenic gene stromal-derived factor-1α (SDF-1α)-an SDF-1α gene-activated scaffold on the functional regulation of human Schwann cells (SCs). A preliminary 2D study was conducted by delivering plasmids encoding for the SDF-1α gene into a monolayer of SCs using polyethyleneimine-based nanoparticles. The delivery of the SDF-1α gene into the SCs enhanced the production of proangiogenic vascular endothelial growth factor (VEGF). Subsequently, we investigated the impact of SDF-1α gene-activated scaffold (3D) on the SCs for 2 weeks, using a gene-free scaffold as control. The transfection of the SCs within the gene-activated scaffold resulted in transient overexpression of SDF-1α transcripts and triggered the production of bioactive VEGF that enhanced endothelial angiogenesis. The overexpression of SDF-1α also caused transient activation of the transcription factor c-Jun and supported the differentiation of SCs towards a repair phenotype. This was characterized by elevated expression of neurotrophin receptor p75NGFR. During this developmental stage, the SCs also performed an extensive remodelling of the basement matrix (fibronectin, collagen IV, and laminin) to enrich their environment with the pro-neurogenic matrix protein laminin, revealing an enhanced pro-neurogenic behavior. Together, this study shows that SDF-1α gene-activated scaffold is a highly bioinstructive scaffold capable of enhancing proangiogenic regenerative response in human SCs for improved wound healing.