One-Pot Extraction of Bioresources from Human Hair via a Zero-Waste Green Route.

In recent years, the extraction of bioresources from biowaste via green chemistry and their utilization for the production of materials has gained global momentum due to growing awareness of the concepts of sustainability. Herein, we report a benign process using an ionic liquid (IL), 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), for the simultaneous extraction of keratin and melanin from human hair. Chemical characterization, secondary structure studies, and thermal analysis of the regenerated protein were performed thoroughly. Hemolytic potential assays demonstrated hemocompatibility of the keratin, and thus, it can be used in blood-contacting biomaterials such as sealants, catheters, hemostats, tissue engineering scaffolds, and so on. Scanning electron microscopy showed retention of the ellipsoidal morphology of melanin after the extraction procedure. The pigment demonstrated the ability to reduce 2,2-diphenyl-1-picrylhydrazyl indicative of its free-radical scavenging activity. Notably, the IL could be recovered and recycled from the dialysis remains which also exhibited conductivity and can be potentially used for bioelectronics. Altogether, this work investigates an extraction process of biopolymers using green chemistry from abundantly available biowaste for the production of biomaterials and does not produce any noxious waste matter.

Direct 3D printing of decellularized matrix embedded composite polycaprolactone scaffolds for cartilage regeneration.

Treatment options for large osteochondral injuries (OCIs) are limited by donor tissue scarcity, morbidity, and anatomic mismatch. 3D printing technology can produce patient-specific scaffolds to address these large defects. Thermoplastics like polycaprolactone (PCL) offer necessary mechanical properties, but lack bioactivity. We fabricated 3D printed PCL scaffolds embedded with polylactic acid microspheres containing decellularized cartilage matrix (DM). DM incorporation within polylactic acid microspheres prevented its thermal degradation during the 3D printing process. The scaffolds replicated the mechanical properties of native cartilage and demonstrated controlled release of DM proteins. Human mesenchymal stem cells (hMSCs) seeded on the composite scaffolds with DM and cultured in basal media self-assembled into aggregates mimicking mesenchymal condensates during embryonic development. The DM composite scaffolds also induced higher expression of biochemical markers of cartilage development than controls, providing evidence for their translational application in the treatment of OCIs. The present study demonstrates the potential of direct incorporation of DM with thermoplastics for 3D printing of patient-specific scaffolds for osteochondral regeneration.

Strategies toward development of antimicrobial biomaterials for dental healthcare applications.

Several approaches for elimination of oral pathogens are being explored at the present time since oral diseases remain prevalent affecting approximately 3.5 billion people worldwide. Need for antimicrobial biomaterials in dental healthcare include but is not restricted to designing resin composites and adhesives for prevention of dental caries. Constant efforts are also being made to develop antimicrobial strategies for clearance of endodontic space prior root canal treatment and for treatment of periimplantitis and periodontitis. This article discusses various conventional and nanotechnology-based strategies to achieve antimicrobial efficacy in dental biomaterials. Recent developments in the design and synthesis of antimicrobial peptides and antifouling zwitterionic polymers to effectively lessen the risks of antimicrobial drug resistance are also outlined in this review. Further, the role of contemporary strategies such as use of smart biomaterials, ionic solvent-based biomaterials and quorum quenchers incorporated biomaterials in the elimination of dental pathogens are described in detail. Lastly, we mentioned the approach of using polymers to print custom-made three-dimensional antibacterial dental products via additive manufacturing technologies. This review provides a critical perspective on the chemical, biomimetic, and engineering strategies intended for developing antimicrobial biomaterials that have the potential to substantially improve the dental health.

Dual Functionalized Injectable Hybrid Extracellular Matrix Hydrogel for Burn Wounds.

Low strength and rapid biodegradability of acellular dermal matrix (ADM) restrict its wider clinical application as a rapid cell delivery platform in situ for management of burn wounds. Herein, the extracted ADM was modified by a dual cross-linking approach with ionic crosslinking using chitosan and covalent cross-linking using an iodine-modified 2,5-dihydro-2,5-dimethoxy-furan cross-linker, termed as CsADM-Cl. In addition, inherent growth factors and cytokines were found to be preserved in CsADM-Cl, irrespective of ionic/covalent crosslinking. CsADM-Cl demonstrated improvement in post crosslinking stiffness with a decreased biodegradation rate. This hybrid crosslinked hydrogel supported adhesion, proliferation, and migration of human foreskin-derived fibroblasts and keratinocytes. Also, the angiogenic potential of CsADM-Cl was manifested by chick chorioallantoic membrane assay. CsADM-Cl showed excellent antibacterial activity against Escherichia coli and Staphylococcus aureus. Moreover, CsADM-Cl treated full thickness burn wounds and demonstrated rapid healing marked with superior angiogenesis, well-defined dermal-epidermal junctions, mature basket weave collagen deposition, and development of more pronounced secondary appendages. Altogether, the bioactive CsADM-Cl hydrogel established significant clinical potential to support wound healing as an apt injectable antibacterial matrix to encounter unmet challenges concerning critical burn wounds.

Bioinspired 3D porous human placental derived extracellular matrix/silk fibroin sponges for accelerated bone regeneration.

Critical bone defects arising from traumatic injury and diseases are of major health concern since they are unable to heal spontaneously without clinical intervention. In this context, bone tissue engineering provides an attractive approach to treat bone defects by providing a bioactive template which has the potential to guide osseous tissue regeneration. In this study, porous hybrid placental extracellular matrix sponge (PIMS) was fabricated by a combinatorial method using silk fibroin (SF)/placental derived extracellular matrix and subsequently evaluated its efficacy towards bone tissue regeneration. The presence of intrinsic growth factors was evidenced by immunoblotting of the extracted proteins derived from the placental derived extracellular matrix. This growth factor rich PIMS lends a unique bioactive scaffolding to human amniotic mesenchymal stem cells (HAMSCs) which supported enhanced proliferation as well as superior osteogenic differentiation. Gene expression studies demonstrated significant up-regulation of osteogenic related genes in the PIMS group. PIMS when implanted in the chick chorioallantoic membrane, significantly attracted allantoic vessels revealing its potential to stimulate angiogenesis ex vivo. Furthermore, no severe immune response to the host was observed on subcutaneous implantation of PIMS in vivo. Instead, it supported the formation of blood vessels, revealing its outstanding biocompatibility. Additionally, critical tibial defects treated with PIMS demonstrated higher bone volume after six weeks when analyzed by micro-CT, which was accompanied by high mineral density. Histological and immunofluorescence studies validated the results and revealed enhanced osseous tissue regeneration after six weeks of surgery. All these findings recapitulated that the growth factors incorporated bioactive PIMS could perform as an appropriate matrix for osteogenic differentiation and efficient bone regeneration.

Novel Process for 3D Printing Decellularized Matrices.

3D bioprinting aims to create custom scaffolds that are biologically active and accommodate the desired size and geometry. A thermoplastic backbone can provide mechanical stability similar to native tissue while biologic agents offer compositional cues to progenitor cells, leading to their migration, proliferation, and differentiation to reconstitute the original tissues/organs1 , 2. Unfortunately, many 3D printing compatible, bioresorbable polymers (such as polylactic acid, PLA) are printed at temperatures of 210 °C or higher - temperatures that are detrimental to biologics. On the other hand, polycaprolactone (PCL), a different type of polyester, is a bioresorbable, 3D printable material that has a gentler printing temperature of 65 °C. Therefore, it was hypothesized that decellularized extracellular matrix (DM) contained within a thermally protective PLA barrier could be printed within PCL filament and remain in its functional conformation. In this work, osteochondral repair was the application for which the hypothesis was tested. As such, porcine cartilage was decellularized and encapsulated in polylactic acid (PLA) microspheres which were then extruded with polycaprolactone (PCL) into filament to produce 3D constructs via fused deposition modeling. The constructs with or without the microspheres (PLA-DM/PCL and PCL(-), respectively) were evaluated for differences in surface features.

Silk Sponges Ornamented with a Placenta-Derived Extracellular Matrix Augment Full-Thickness Cutaneous Wound Healing by Stimulating Neovascularization and Cellular Migration.

Regeneration of full-thickness wounds without scar formation is a multifaceted process, which depends on in situ dynamic interactions between the tissue-engineered skin substitutes and a newly formed reparative tissue. However, the majority of the tissue-engineered skin substitutes used so far in full-thickness wound healing cannot mimic the natural extracellular matrix (ECM) complexity and thus are incapable of providing a suitable niche for endogenous tissue repair. Herein, we demonstrated a simple approach to fabricate porous hybrid ECM sponges (HEMS) using a placental ECM and silk fibroin for full-thickness wound healing. HEMS with retained cytokines/growth factors provided a noncytotoxic environment in vitro for human foreskin fibroblasts (HFFs), human epidermal keratinocytes (HEKs), and human amniotic membrane-derived stem cells to adhere, infiltrate, and proliferate. Interestingly, HEMS-conditioned media accelerated the migration of HFFs and HEKs owing to the presence of cytokines/growth factors. Also, the ex vivo chick chorioallantoic membrane assay of HEMS demonstrated its excellent vascularization potential by inducing and supporting blood vessels. Additionally, HEMS when subcutaneously implanted demonstrated no severe immune response to the host. Furthermore, HEMS implanted in full-thickness wounds in a rat model showed augmented healing progression with well-organized epidermal-dermal junctions via pronounced angiogenesis, accelerated migration of HFFs/HEKs, enhanced granulation tissue formation, and early re-epithelialization. Taken together, these findings show that porous HEMS ornamented with cytokines/growth factors having superior physicomechanical properties may be an appropriate skin substitute for full-thickness cutaneous wounds.

Microspheres containing decellularized cartilage induce chondrogenesis in vitro and remain functional after incorporation within a poly(caprolactone) filament useful for fabricating a 3D scaffold.

In this study, articular cartilage was decellularized preserving a majority of the inherent proteins, cytokines, growth factors and sGAGs. The decellularized cartilage matrix (dCM) was then encapsulated in poly(lactic acid) microspheres (MS + dCM) via double emulsion. Blank microspheres without dCM, MS(-), were also produced. The microspheres were spherical in shape and protein encapsulation efficiency within MS + dCM was 63.4%. The sustained release of proteins from MS + dCM was observed over 4 weeks in vitro. Both MS + dCM and MS(-) were cytocompatible. The sustained delivery of retained growth factors and cytokines from MS + dCM promoted cell migration in contrast to MS(-). Subsequently, chondrogenesis of human mesenchymal stem cells was upregulated in presence of MS + dCM as evidenced from immunohistochemistry, biochemical quantification and qPCR studies. Specifically, collagen II, aggrecan and SOX 9 gene expression were increased in the presence of MS + dCM by an order or more in magnitude compared to MS(-) with concomitant downregulation of hypertrophic genes (COL X) despite being cultured in the absence of chondrogenic media, (p < 0.05). Lastly, microspheres containing alkaline phosphatase (MS + ALP), a surrogate to assess the thermal stability of dCM proteins, incorporated within poly(caprolactone) filaments showed that the enzyme remained functional after filament production by melt extrusion. The establishment of a novel, thermally stable process for producing filaments containing chondroinductive microspheres provides evidence supporting subsequent development of a clinically-relevant, 3D scaffold fabricated from them for osteochondral regeneration and repair.

Biocompatible nanogel derived from functionalized dextrin for targeted delivery of doxorubicin hydrochloride to MG 63 cancer cells.

The present article demonstrates the targeted delivery of doxorubicin hydrochloride to human osteosarcoma cancer cell lines (MG 63) using functionalized dextrin based crosslinked, pH responsive and biocompatible nanogel. The nanogel has been prepared through Michael-type addition reaction using dextrin (Dxt), N, N'-methylene bisacrylamide (MBA, as crosslinker), acrylic acid (AA, as monomer) and potassium persulfate (KPS, as initiator). The structure, composition, morphology of the nanogel have been explored using FTIR and 1H NMR spectroscopy, XRD, TGA, DSC, CHN and AFM analyses. The TEM analysis confirmed that the size of nanogel appeared within 100nm, while DLS study indicates that the diameter of the nanogel remained between 113 and 126nm. The AFM study implied the porous morphology of the synthesized nanogel. The rheological study suggests the gel behaviour of the synthesized nanogel at 37±0.1°C. Difference in% swelling at pH 5.5 and 7.4 indicates pH-responsiveness of the nanogel. The in vitro cytocompatibility results ascertained that the nanogel is non-toxic to human mesenchymal stem cells (hMSCs). In vitro cellular uptake study confirmed that FITC-loaded nanogel can cross the cellular membrane and be well uptake by the cell cytoplasm. The nanogel could efficiently encapsulate doxorubicin hydrochloride (Dox) with the loading efficiency of 27±0.2% after 72h. The Dox-loaded nanogel demonstrates anti-cancer activity towards MG 63 cancer cells and release the encapsulated drug in a controlled way.

Investigating the potential of human placenta-derived extracellular matrix sponges coupled with amniotic membrane-derived stem cells for osteochondral tissue engineering.

Osteochondral injuries are challenging to repair due to their complex tissue anatomy and restricted self-repairing ability associated with a limited blood supply. Osteochondral tissue engineering is an important clinical aspect of the management and treatment of cartilage and underlying bone. In the present study, we fabricated human placenta-derived extracellular matrix sponges (PEMS) for repair of osteochondral tissue through a decellularization process. There were no significant cellular components present in the PEMS; hematoxylin & eosin/DAPI staining, DNA quantification and agarose gel electrophoresis were used to evaluate the extent of decellularization. Moreover, no significant alteration to the collagen and glycosaminoglycan (native extracellular matrix) content of the PEMS was observed. PEMS in vitro provided a non-cytotoxic environment rich in bioactive cues for human amniotic membrane-derived stem cells (HAMSCs) to proliferate in and differentiate into chondrogenic and osteogenic lineages under induction. Histological analysis at 28 days after the PEMS were subcutaneously implanted demonstrated no severe immune response in the host and supported the formation of blood vessels. To assess the osteochondral tissue repair ability of PEMS, cell-free PEMS (CFP) and cell-seeded PEMS (CSP) were implanted at osteochondral defect sites in a rabbit model. Histological scores indicated that osteochondral regeneration was more successful in the defects filled with CSP compared to those filled with CFP and empty defects (ED) after 60 days of implantation. In summary, a naturally derived biocompatible scaffold composed of extracellular matrix from human placenta has been successfully developed for osteochondral tissue engineering.

In Situ Iodination Cross-Linking of Silk for Radio-Opaque Antimicrobial Surgical Sutures.

The purpose of the study is to develop a method of imparting radio-opacity to the silk fibers by stepwise 2,5-dimethoxy-2,5-dihydro-furan (DMDF)-iodine cross-linking reaction for suture fabrication with mechanical properties abiding with U.S. pharmacopeia guidelines along with non invasive imaging advantage in postoperative follow-up. Silk fibers isolated from Bombyx mori were cross-linked with suitable concentration of DMDF linked with iodine under elevated temperature and pressure. Cross-linked fibers knitted into sutures were subjected to further testing.Computed tomography (CT) images on day 28 of in vivo studies showed mean radio-opacity value (MRV) of 213 ± 19.46 compared to the vertebral bone having value of 254.66 ± 0.51. Modified silk sutures demonstrated several advantages like high tensile strength (626 ± 23.3 MPa) and knot strength (388.6 ± 16.8 MPa) besides antimicrobial property. Encouraging preliminary in vitro and in vivo biocompatibility studies advocate the potential use of modified suture material in cardiac surgery, aneurysmal embolization surgeries, and arterio-venous occlusion surgeries.

Stimulus-Responsive, Biodegradable, Biocompatible, Covalently Cross-Linked Hydrogel Based on Dextrin and Poly(N-isopropylacrylamide) for in Vitro/in Vivo Controlled Drug Release.

A novel stimulus-sensitive covalently cross-linked hydrogel derived from dextrin, N-isopropylacrylamide, and N,N'-methylene bis(acrylamide) (c-Dxt/pNIPAm), has been synthesized via Michael type addition reaction for controlled drug release application. The chemical structure of c-Dxt/pNIPAm has been confirmed through Fourier transform infrared (FTIR) spectroscopy and (1)H and (13)C NMR spectral analyses. The surface morphology of the hydrogel has been studied by field emission scanning electron microscopic (FE-SEM) and environmental scanning electron microscopic (E-SEM) analyses. The stimulus responsiveness of the hydrogel was studied through equilibrium swelling in various pH media at 25 and 37 °C. Rheological study was performed to measure the gel strength and gelation time. Noncytotoxicity of c-Dxt/pNIPAm hydrogel has been studied using human mesenchymal stem cells (hMSCs). The biodegradability of c-Dxt/pNIPAm was confirmed using hen egg lysozyme. The in vitro and in vivo release studies of ornidazole and ciprofloxacin imply that c-Dxt/pNIPAm delivers both drugs in a controlled way and would be an excellent alternative for a dual drug carrier. The FTIR, powder X-ray diffraction (XRD), and UV-vis-near infrared (NIR) spectra along with the computational study predict that the drugs remain in the matrix through physical interaction. A stability study signifies that the drugs (ornidazole ∼97% and ciprofloxacin ∼98%) are stable in the tablet formulations for up to 3 months.

Dextrin and poly(acrylic acid)-based biodegradable, non-cytotoxic, chemically cross-linked hydrogel for sustained release of ornidazole and ciprofloxacin.

Herein, novel biodegradable, stimulus-responsive, chemically cross-linked and porous hydrogel has been synthesized to evaluate its applicability as an efficient carrier for sustained release of ornidazole and ciprofloxacin. The cross-linked hydrogel (c-Dxt/pAA) has been developed from dextrin and poly(acrylic acid) using N,N'-methylene bis(acrylamide) cross-linker via Michael-type addition reaction. With the variation of reaction parameters, various c-Dxt/pAA hydrogels have been synthesized to optimize the best one. c-Dxt/pAA hydrogel has been characterized using various physicochemical characterization techniques. The hydrogel demonstrates significant pH and temperature sensitivity. Gel characteristics and gel kinetics have been performed through the measurement of rheological parameters. The hydrogel shows noncytotoxic behavior toward human mesenchymal stem cells. Biodegradation study predicts that c-Dxt/pAA is degradable in nature. The in vitro release of ornidazole and ciprofloxacin suggests that the hydrogel released both the drugs in a controlled manner with extensive stability up to 3 months. The results suggest that c-Dxt/pAA is probably a promising candidate for controlled release of ornidazole and ciprofloxacin.

Covalent cross-links in polyampholytic chitosan fibers enhances bone regeneration in a rabbit model.

Chitosan fibers were prepared in citric acid bath, pH 7.4 and NaOH solution at pH 13, to form ionotropically cross-linked and uncross-linked fibers, respectively. The fibers formed in citric acid bath were further cross-linked via carbodiimide chemistry; wherein the pendant carboxyl moieties of citric acid were used for new amide bond formation. Moreover, upon covalent cross-linking in the ionically gelled citrate-chitosan fibers, incomplete conversion of the ion pairs to amide linkages took place resulting in the formation of a dual network structure. The dual cross-linked fibers displayed improved mechanical property, higher stability against enzymatic degradation, hydrophobicity and superior bio-mineralization compared to the uncross-linked and native citrate cross-linked fibers. Additionally, upon cyclic loading, the ion pairs in the dual cross-linked fibers dissociated by dissipating energy and reformed during the relaxation period. The twin property of elasticity and energy dissipation mechanism makes the dual cross-linked fiber unique under dynamic mechanical conditions. The differences in the physico-chemical characteristics were reflected in protein adsorption, which in turn influenced the cellular activities on the fibers. Compared to the uncross-linked and ionotropically cross-linked fibers, the dual cross-linked fibers demonstrated higher proliferation and osteogenic differentiation of the MSCs in vitro as well as better osseous tissue regeneration in a rabbit model.

Chitosan derivatives cross-linked with iodinated 2,5-dimethoxy-2,5-dihydrofuran for non-invasive imaging.

Radiopaque polymer derivatives were successfully prepared through surface diffusion mediated cross-linking of chitosan with iodinated 2,5-dimethoxy-2,5-dihydrofuran. The incorporation of iodine in 2,5-dimethoxy-2,5-dihydrofuran was validated by (1)H NMR and mass spectroscopy. The cross-linking of the glucosamine moieties of chitosan with the iodinated product was confirmed by (13)C NMR and energy-dispersive X-ray spectroscopy. Radiography analysis proved inherent opacity of the iodinated fibrous sheets and microspheres that were comparable to the X-ray visibility of aluminum hollow rings of equivalent thickness and commercially available radiopaque tape, respectively. Microscopic studies evidenced retention of the fiber/microsphere morphology after the iodination/cross-linking reactions. The effects of iodination/cross-linking on the mechanical and biodegradation properties of fibers were studied by nanoindentation and enzymatic assay, respectively. In vitro and in vivo studies established the nontoxic, biodegradable nature of radiopaque derivatives. Iodinated fiber mesh implanted in a rabbit model was significantly X-ray opaque compared to the uncross-linked fiber mesh and medical grade surgical swabs. Further, opacity of the iodinated mesh was evident even after 60 days, though the intensity was reduced, which indicates the biodegradable nature of the iodinated polymer. The opacity of the iodinated sutures was also established in the computed tomography images. Finally, the sufficient in vivo contrast property of the radiopaque microspheres in the gastrointestinal tract indicates its possible role in clinical diagnostics.

Citrate cross-linked gels with strain reversibility and viscoelastic behavior accelerate healing of osteochondral defects in a rabbit model.

Most living tissues are viscoelastic in nature. Self-repair due to the dissipation of energy by reversible bonds prevents the rupture of the molecular backbone in these tissues. Recent studies, therefore, have aimed to synthesize biomaterials that approximate the mechanical performance of biological materials with self-recovery properties. We report an environmentally friendly method for the development of ionotropically cross-linked viscoelastic chitosan gels with a modulus comparable to that of living tissues. The strain recovery property was found to be highest for the gels with the lowest cross-linking density. The force-displacement curve showed significant hysteresis due to the presence of reversible bonds in the cross-linked gels. Nanoindentation studies demonstrated the creep phenomenon for the cross-linked chitosan gels. Creep, hysteresis, and plasticity index confirmed the viscoelastic behavior of the cross-linked gels. The viscoelastic gels were implanted at osteochondral defect sites to assess the tissue regeneration ability. In vivo results demonstrated early cartilage formation and woven bone deposition for defects filled with the gels compared to nontreated defects.

In vitro ALP and osteocalcin gene expression analysis and in vivo biocompatibility of N-methylene phosphonic chitosan nanofibers for bone regeneration.

Most polymeric nanofibers used for bone tissue engineering lack adequate functional groups for bioactivity. This study explores the potential of nanofibers of phosphate functionalized derivative of chitosan-N-methylene phosphonic chitosan (NMPC) for bone tissue engineering. Nanofibers were fabricated by electrospinning of NMPC/PVA blend solutions. NMPC/PVA nanofibers exhibited 172% higher viability of MG-63 cells compared to pure PVA nanofibers. ALP and Collagen type I genes revealed higher expression in NMPC nanofibers on day 3 whereas osteocalcin gene was expressed on day 7. In rabbit tibial defects, NMPC based electrospun graft showed presence of no adverse tissue reaction by histological examination while radiological examination suggested acceleration of bone healing by 300% compared to defects without any scaffold. Thus it is concluded NMPC based nanofibers may have potential for bone grafting applications.