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Scar formation is a common consequence of skin injuries that lead to a wide range of adverse effects including physical deformities and psychological disorders. Studies demonstrate that mammalian fetal skin in early gestation is capable of regenerating itself after injury without any scar formation. A number of potential therapies have been developed to reduce scar formation in cutaneous wounds based on differences between the process of adult and fetal wound healing. The ideal approach to eliminate scar formation after skin injury is to use a pro-regenerative matrix along with growth factors and cell types that induce regeneration rather than repair. This work provides a comprehensive review of engineering approaches to scar-free wound healing with emphasis on the use of pro-regenerative biomaterials to minimize scar formation in skin injuries. IMPACT STATEMENT: Millions of people every year develop scars in response to skin injuries after surgery, trauma, or burns with significant undesired physical and psychological effects. This review provides an update on engineering strategies for scar-free wound healing and discusses the role of different cell types, growth factors, cytokines, and extracellular components in regenerative wound healing. The use of pro-regenerative matrices combined with engineered cells with less intrinsic potential for fibrogenesis is a promising strategy for achieving scar-free skin tissue regeneration.

Engineering approaches that mimic the process of fetal development have the potential to regenerate the zonal organization of articular cartilage. The objective of this study was to investigate the effect of sequential addition of zone-specific growth factors such as bone morphogenetic protein (BMP)-7, insulin-like growth factor (IGF)-1, and Indian hedgehog (IHH) to transforming growth factor (TGF)-β1-supplemented chondrogenic medium on zonal differentiation of human mesenchymal stem cells (hMSCs) encapsulated in an articular cartilage-derived matrix. First, fetal or adult bovine articular cartilage was decellularized, digested, and methacrylate functionalized to produce an injectable macromer (CarMa, f-CarMa for fetal, a-CarMa for adult) for encapsulation of hMSCs. Next, the optimum matrix source and initial cell density for chondrogenic differentiation of hMSCs to the superficial and calcified zone phenotypes were determined by encapsulation of the cells in CarMa hydrogel and incubated in chondrogenic medium/TGF-β1 supplemented with BMP-7 and IHH, respectively. Then, the encapsulated hMSCs were preexposed to BMP-7-supplemented chondrogenic medium/TGF-β1 and the effect of sequential addition of IGF-1 and IHH to the medium on the expression of zone-specific markers was investigated. According to the results, f-CarMa and high cell density enhanced differentiation of the encapsulated hMSCs to the superficial zone phenotype, whereas a-CarMa and low cell density enhanced differentiation to the calcified zone. The addition of IGF-1 to the chondrogenic medium/TGF-β1 stimulated differentiation of the encapsulated hMSCs, preexposed to BMP-7, to the middle zone phenotype. The addition of IHH to the chondrogenic medium/TGF-β1 stimulated maturation of the encapsulated hMSCs, preexposed to BMP-7 and IGF-1, to the calcified zone phenotype. The results are potentially useful for engineering injectable, cellular hydrogels for regeneration of full-thickness articular cartilage. IMPACT STATEMENT: The higher regenerative capacity of fetal articular cartilage compared with the adult is rooted in differences in cell density and matrix composition. We hypothesized that the zonal organization of articular cartilage can be engineered by encapsulation of mesenchymal stem cells in a single superficial zone-like matrix followed by sequential addition of zone-specific growth factors within the matrix, similar to the process of fetal cartilage development. The results demonstrate that the zonal organization of articular cartilage can potentially be regenerated using an injectable, monolayer cell-laden hydrogel with sequential release of growth factors.

The objective of this work was to engineer self-assembled nanoparticles (NPs) for on-demand release of bone morphogenetic protein-2 (BMP2) and vascular endothelial growth factor (VEGF) in response to enzymes secreted by the migrating human mesenchymal stem cells (hMSCs) and human endothelial colony forming cells (ECFCs) to induce osteogenesis and vasculogenesis. Gene expression profiling experiments revealed that hMSCs and ECFCs, encapsulated in osteogenic/vasculogenic hydrogels, expressed considerable levels of plasminogen, urokinase plasminogen activator and its receptor uPAR, and tissue plasminogen activator. Therefore, the plasmin-cleavable lysine-phenylalanine-lysine-threonine (KFKT) was used to generate enzymatically cleavable NPs. The acetyl-terminated, self-assembling peptide glycine-(phenylalanine)3GFFF-ac and the plasmin-cleavable GGKFKTGG were reacted with the cysteine-terminated CGGK(Fmoc/MTT) peptide through the MTT and Fmoc termini, respectively. The difunctional peptide was conjugated to polyethylene glycol diacrylate (PEGDA) with molecular weights (MW) ranging from 0.5 to 7.5 kDa, and the chain ends of the PEG-peptide conjugate were terminated with succinimide groups. After self-assembly in aqueous solution, BMP2 was grafted to the self-assembled, plasmin-cleavable PEG-based (PxSPCP) NPs for on-demand release. The NPs' stability in aqueous solution and that of the grafted BMP2 were strongly dependent on PEG MW. P2SPCP NPs showed high particle size stability, BMP2 grafting efficiency, grafted protein stability, and high extent of osteogenic differentiation of hMSCs. The localized and on-demand release of BMP2 from PxSPCP NPs coencapsulated with hMSCs in the linear polyethylene glycol-co-lactide acrylate patterned hydrogel with microchannels encapsulating hMSCs + ECFCs and VEGF-conjugated nanogels resulted in the highest extent of osteogenic and vasculogenic differentiation of the encapsulated cells compared to directly added BMP2/VEGF. The on-demand release of BMP2 from PxSPCP NPs not only enhances osteogenesis and vasculogenesis but also potentially reduces many undesired side effects of BMP2 therapy in bone regeneration.

The objective of this work was to fabricate a rigid, resorbable and osteoconductive scaffold by mimicking the hierarchical structure of the cortical bone. Aligned peptide-functionalize nanofiber microsheets were generated with calcium phosphate (CaP) content similar to that of the natural cortical bone. Next, the CaP-rich fibrous microsheets were wrapped around a microneedle to form a laminated microtube mimicking the structure of an osteon. Then, a set of the osteon-mimetic microtubes were assembled around a solid rod and the assembly was annealed to fuse the microtubes and form a shell. Next, an array of circular microholes were drilled on the outer surface of the shell to generate a cortical bone-like scaffold with an interconnected network of Haversian- and Volkmann-like microcanals. The CaP content, porosity and density of the bone-mimetic microsheets were 240 wt%, 8% and 1.9 g/ml, respectively, which were close to that of natural cortical bone. The interconnected network of microcanals in the fused microtubes increased permeability of a model protein in the scaffold. The cortical scaffold induced osteogenesis and vasculogenesis in the absence of bone morphogenetic proteins upon seeding with human mesenchymal stem cells and endothelial colony-forming cells. The localized and timed-release of morphogenetic factors significantly increased the extent of osteogenic and vasculogenic differentiation of human mesenchymal stem cells and endothelial colony-forming cells in the cortical scaffold. The cortical bone-mimetic nature of the cellular construct provided balanced rigidity, resorption rate, osteoconductivity and nutrient diffusivity to support vascularization and osteogenesis.

The present communication reports on changes in the secondary and tertiary structures of Hemoglobin (Hb) upon fructation. The presence of Lemon balm (LB) provided significant protection toward the formation of early (HbA1c) and advanced glycation end products (Hb-AGEs), as evident from circular dichroism (CD) and fluorescence studies. The degree of AGE modification and its inhibition were characterized with intrinsic fluorescence, the fibrillar state, and the exposure of hydrophobic clusters. An increased signal of intrinsic fluorescence was observed upon glycation, particularly in the later stage of fructation. Glycation mediated fibril formation and its inhibition were assessed by Thioflavin T (ThT) assay. These sequence of events were parallel with an increase in β-sheet content from ~2 to 13% for Hb-AGE, as evidenced by Congo red binding and CD. Furthermore, the late stage of glycation was also marked by an increase loss of heme moiety, confirming the relatively high affinity of ANS toward glycated-globin which was mitigated by applying the balm extract. However, the activity of balm can be attributed to inhibition of oxidative stress and free radicals derived from Hb-AGE, or chelation of metal ions producing in Fenton reaction. Overall, the presence of LB provided significant protection against AGE-induced deleterious processes, can qualify the herb as an effective AGE-inhibitor with potential prevention toward diabetic complications arising from Hb glycation.

The objective of this work was to investigate the effect of devitalized human mesenchymal stem cells (hMSCs) and endothelial colony-forming cells (ECFCs) seeded on mineralized nanofiber microsheets on protein release, osteogenesis, vasculogenesis, and macrophage polarization. Calcium phosphate nanocrystals were grown on the surface of aligned, functionalized nanofiber microsheets. The microsheets were seeded with hMSCs, ECFCs, or a mixture of hMSCs+ECFCs, cultured for cell attachment, differentiated to the osteogenic or vasculogenic lineage, and devitalized by lyophilization. The release kinetic of total protein, bone morphogenetic protein-2 (BMP2), and vascular endothelial growth factor (VEGF) from the devitalized microsheets was measured. Next, hMSCs and/or ECFCs were seeded on the devitalized cell microsheets and cultured in the absence of osteo-/vasculo-inductive factors to determine the effect of devitalized cell microsheets on hMSC/ECFC differentiation. Human macrophages were seeded on the microsheets to determine the effect of devitalized cells on macrophage polarization. Based on the results, devitalized undifferentiated hMSC and vasculogenic-differentiated ECFC microsheets had highest sustained release of BMP2 and VEGF, respectively. The devitalized hMSC microsheets did not affect M2 macrophage polarization while vascular-differentiated, devitalized ECFC microsheets did not affect M1 polarization. Both groups stimulated higher M2 macrophage polarization compared to M1.