Keratin is an insoluble filamentous sulphur-rich protein constituting the bulk of epidermal appendages, such as hair, chicken feathers, nails, and claws. Being ubiquitously present, keratin's efficacy has been extensively evaluated for different tissue engineering applications. However, to date, no systematic study has investigated and compared two conformationally different keratins sourced from human hair and chicken feathers. The present research emphasizes the comparative therapeutic efficacy of keratins derived from human hair and chicken feathers. Herein, keratins isolated from both sources revealed differences in their molecular weights, as assessed using SDS-PAGE analysis. Notably, human hair-derived keratin (HK) has a molecular weight of 40-60 kDa, while chicken feather-derived keratin (CK) has a molecular weight of ∼10 kDa. FTIR, CD and Raman spectroscopy were performed to assess their conformational features, evidencing the α-helix dominance in HK but β-sheet/turn dominance in CK. Immunological assessment of the macrophage cell line demonstrated the anti-inflammatory property of keratin from each source, where HK showed a significantly higher anti-inflammatory property than CK. Furthermore, in vivo assessment showed that HK-treated wounds exhibit enhanced re-epithelialization and balanced COLI/COLIII deposition, indicating minimal scar formation compared with CK-treated ones. These findings underscore the potential of HK in clinical applications, particularly in wound care and regenerative medicines.

Being an excretory scleroprotein, human hair-derived keratin with inherent bioactive peptide cues may actively participate in an immunomodulatory role in the wound microenvironment. In the current study, nano-microfibrous structural attributes mimicking the extracellular matrix were prepared using a polymer blend containing a high loading of keratin as a bioactive matrix by electrospinning, where polycaprolactone (PCL) was used as an electrospinnable aid. The FESEM analysis showed smooth fibers with diameters ranging from 100 to 220 nm. High keratin loading facilitated improved cellular affinity due to the presence of bioactive peptide cues. Physico-chemical characterization confirmed the presence of protein within the PCL matrix, and the modulus of the material (~25 MPa) was found to be similar to that of native skin. Furthermore, keratin-rich matrices evidenced the potential to modulate macrophages toward M2 macrophages. In vitro assessment with human dermal fibroblasts (HDFs) demonstrated enhanced cytocompatibility-like cellular activity and cell proliferation. In vivo studies evidenced the proactive role of the KPCL matrix in supporting full-thickness wound healing and balancing macrophage activity (CD68 and CD206 immunostaining). Immunohistochemistry and RT-PCR studies showed increased COLI and COLIII expression, evidencing dermal reconstruction within 18 days. Enhanced P63 and K14 expression supported the synergistic role of reepithelialization by the matrix enriched with keratin. Overall, the study showed that the keratin-based matrix facilitates skin wound healing.

The quest for an ideal wound dressing material has been a strong motivation for researchers to explore novel biomaterials for this purpose. Such explorations have led to the extensive use of silk fibroin (SF) as a suitable polymer for several applications over the years. Unfortunately, another major silk protein—sericin has not received its due attention yet in spite of having favorable biological properties. In this study, we report an approach of blending SF and silk sericin (SS) without the usage of chemical crosslinkers is made possible by the usage of formic acid which evaporates to induce β -sheets formation to form cytocompatible films. Raman spectroscopy confirms the presence of SF/SS components in blend and formation of β -sheet in films. In situ , gelation kinetics studies were conducted to understand the change in gelation properties with addition of sericin into SF. Methyl thiazolyl tetrazolium and live/dead assays were performed to study cellular attachment, viability and proliferation on SF/SS films. The antibacterial properties of SF/SS films were tested using Gram-negative and Gram-positive bacteria. The re-structured SF/SS films were stable, transparent, show good mechanical properties, antibacterial activity and cytocompatibility, therefore can serve as suitable biomaterial candidates for skin regeneration applications.

Chronic wounds suffer from impaired healing due to microbial attack and poor vascular growth. Thermoresponsive hydrogels gained attention in wound dressing owing to their gelation at physiological temperature enabling them to take the shape of asymmetric wounds. The present study delineates the development of thermoresponsive hydrogel (MCK), from hair-derived keratin (K) and methylcellulose (MC) in the presence of sodium sulfate. The gelation temperature (Tg) of this hydrogel is in the range of 30 °C to 33 °C. Protein-polymer interaction leading to thermoreversible sol-gel transition involved in MCK blends has been analyzed and confirmed by FTIR, XRD, and thermal studies. Keratin, has introduced antioxidant properties to the hydrogel imparted cytocompatibility towards human dermal fibroblasts (HDFs) as evidenced by both MTT and live dead assays. In vitro wound healing assessment has been shown by enhanced migration of HDFs in the presence of MCK hydrogel compared to the control. Also, CAM assay and CD31 expression by the Wistar rat model has shown increased blood vessel branching after the implantation of MCK hydrogel. Further, in vivo study, demonstrated MCK efficacy of hydrogel in accelerating full-thickness wounds with minimal scarring in Wistar rats, re-epithelialization, and reinstatement of the epidermal-dermal junction thereby exhibiting clinical relevance for chronic wounds.

Antibiotic-impregnated bioactive therapeutic biomaterials are highly demanding due to the risk of infection associated with musculoskeletal injuries and bone replacement surgeries. In this study, we report the synthesis of antibiotic nanohybrid having an aqueous sensitive sheddable polycaprolactone (PCL) shell grafted over gentamicin sulphate (GS)-charged bioactive glass (BG) nanospheres via surface-initiated polymerization. This study indeed shows the additional role of GS, while its potential as an initiator for the ring opening polymerization of ε-caprolactone (CL) is investigated by choosing 1:0.175 and 1:0.53 weight ratio of CL:GS-loaded nanoparticles. The influence of monomer/initiator feed ratio on the synthesized PCL was evident in the unbound PCL fractions, suggesting the effective role of GS as an initiator. However, in the nanohybrids, the percentage grafting and shell thickness were comparable regardless of the quantity of drug-loaded particles used. The 1H NMR and MALDI-ToF analysis of the unbound PCL further confirmed the part of GS with the hydroxyl end group served as the surface anchoring group. The successful grafting of PCL was confirmed by different characterization techniques. The effective shedding of the shell in the aqueous environment and the subsequent release of GS were confirmed by the TEM analysis and zeta potential measurements. Additionally, the drug release was modulated with controlled burst release with only 15 wt% and 14 wt% release in the nanohybrids in the first 8 h, while it is 29 wt% for the unmodified BG nanoparticles. Studies of growth inhibition of clinically relevant strains such as E.coli, S.aureus, P.aeruginosa and B.subtilis indicated that the GS released from the nanohybrid retained antibacterial activity. Thus the GS-initiated polyester functionalization of drug-loaded BG nanoparticles, which enables an environment responsive as well as modulated release, is considered beneficial for further progresses in drug-loaded scaffolds.

Targeted delivery of site-specific therapeutic agents is an effective strategy for osteoarthritis treatment. The lack of blood vessels in cartilage makes it difficult to deliver therapeutic agents like peptides to the defect area. Therefore, nucleus-targeting zwitterionic carbon nano-dots (CDs) have immense potential as a delivery vehicle for effective peptide delivery to the cytoplasm as well as nucleus. In the present study, nucleus-targeting zwitterionic CDs have been synthesized as delivery vehicle for peptides while also working as nano-agents towards optical monitoring of cartilage healing. The functional groups of zwitterion CDs were introduced by a single-step microwave assisted oxidation procedure followed by COL II peptide conjugation derived from Capra auricular cartilage through NHS/EDC coupling. The peptide-conjugated CDs (PCDs) allows cytoplasmic uptake within a short period of time (∼30 m) followed by translocation to nucleus after ∼24 h. Moreover, multicolor fluorescence of PCDs improves (blue, green, and read channel) its sensitivity as an optical code providing a compelling solution towards enhanced non-invasive tracking system with multifunctional properties. The PCDs-based delivery system developed in this study has exhibited superior ability to induce ex-vivo chondrogenic differentiation of ADMSCs as compared to bare CDs. For assessment of cartilage regeneration potential, pluronic F-127 based PCDs hydrogel was injected to rabbit auricular cartilage defects and potential healing was observed after 60 days. Therefore, the results confirm that PCDs could be an ideal alternate for multimodal therapeutic agents.

Natural materials derived/extracted Ceramics is an excellent material for developing ceramic-based orthopedic implants. Recently, we have demonstrated an easily scalable, energy-efficient green method to extract ceramic particles from bio-waste i.e. chicken bone. Though the chicken bone extract (CBE) has good biocompatibility, it lacks good mechanical properties in the 3D printed condition as that of human bones. Here, we have reinforced CBE with different weight proportions of silicon carbide to improve the mechanical characteristics of the composite. The hybrid of CBE (oxide) and carbide (SiC) is sintered at different temperatures to understand the effect of the interface of the two ceramics. It is observed that temperature has minimal effect and composition has a noticeable effect on mechanical strength as well as bio-toxicity. The toughness (∼3.58 MJ/m3) and compressive strength (∼64.64 MPa) of the 90:10 composition sintered at 1250 °C show the maximum optimum values. A mathematical model has also been developed to predict and correlate the toughness with porosity, volumetric loading, and elastic modulus of the 3D-printed ceramic composite.

Chronic wounds are the outcome of an imbalanced inflammatory response caused by sustenance of immune microenvironment. In this context, tissue engineered graft played great role in healing wounds but faced difficulty in scar remodelling, immune rejection and poor vascularization. All the limitations faced are somewhere linked with the immune cells involved in healing. In this consideration, immunomodulatory biomaterials bridge a large gap with the delivery of modulating factors for triggering key inflammatory cells responsible towards interplay in the wound micro-environment. Inherent physico-chemical properties of biomaterials substantially determine the nature of cell-materials interaction thereby facilitating differential cytokine gradient involved in activation or suppression of inflammatory signalling pathways, and followed by surface marker expression. This review aims to systematically describe the interplay of immune cells involved in different phases in the wound microenvironment and biomaterials. Additionally, it also focuses on modulating innate immune cell responses in the context of triggering the halted phase of the wound healing, i.e., inflammatory phase. The various strategies are highlighted for modulation of wound microenvironment towards wound regeneration including stem cells, cytokines, growth factors, vitamins, and anti-inflammatory agents to induce interactive ability of biomaterials with immune cells. The last section focuses on prospective approaches and current potential strategies for wound regeneration. This includes the development of different models to bridge the gap between mouse models and human patients. Emerging new tools to study inflammatory response owing to biomaterials and novel strategies for modulation of monocyte and macrophage behaviour in the wound environment are also discussed.

With the fast changing lifestyle, vitamin D deficiency is becoming extremely common. Therefore, development of economical, efficient, and fast sensors for vitamin D is the need of the hour. Carbon-based nanomaterials are extensively explored in sensing of variety of biomolecules. In the present study, an antibody-free, highly sensitive, carbon-nanotube-based, highly responsive vitamin D3 sensor is reported. Nitrogen-doped carbon nanotubes are utilized to overcome the limiting factor of hydrophobic character of pure carbon. The synthesized N-doped CNTs showed a specific surface area of 24 m2/g. The surface charges of vitamin D3 and the vitamin D3/NCNT complex are found to be -20 and -6.4 mV, respectively, by zeta potential measurements. The sensor is able to deliver high performance in the concentration range of 0-10 nM, with a limit of detection of 16 pM. The response study indicated the sensitivity value as 0.000495 mA/cm2 nM. The sensor is also able to show a higher selectivity toward vitamin D3 in comparison to other biomolecules. The long-term stability, reproducibility, good linear range, and ultralow detection capability of the sensor are also reported.

In the realm of diagnostics and emerging therapeutics, healthcare sectors like therapeutics, point-of-care diagnostics (POCT) and theranostics have been witnessing a paradigm shift. The complexity of life-style in present scenario demands sophisticated and dedicated medical devices that can be deployed for obtaining desired outcomes both in quick succession and also impart long-term benefits. The advent of such emerging healthcare devices will immensely impact patient health especially when the turn-around time is critical, chronic injuries, burn patients, cardiac arrest or brain injury being some of the examples for such conditions. It is of paramount importance to highlight that a well-engineered platform is the core component of any therapeutic system. Designing platforms with multidimensional functionality provides a stable microenvironment, capable of mimicking the dynamic in vivo conditions thus securing the success aspect of any medical device. The interaction of cells, tissues and biomolecules with its microenvironment involve sequential coherence of complex biochemical events leading to occurrence of desired bio-affinitive reactions. For a heterogeneous system, bio-affinitive reactions demands precise control over the microenvironment for specific therapeutic applications as fate of biomolecules depends on their surroundings and may face steric hindrance and easily lose their activity when brought in close proximity to such platforms.

Tryptophan-rich antigens play important role in host-parasite interaction. One of the Plasmodium vivax tryptophan-rich antigens called PvTRAg33.5 had earlier been shown to be predominantly of alpha helical in nature with multidomain structure, induced immune responses in humans, binds to host erythrocytes, and its sequence is highly conserved in the parasite population. In the present study, we divided this protein into three different parts i.e. N-terminal (amino acid position 24-106), middle (amino acid position 107-192), and C-terminal region (amino acid position 185-275) and determined the erythrocyte binding activity of these fragments. This binding activity was retained by the middle and C-terminal fragments covering 107 to 275 amino acid region of the PvTRAg33.5 protein. Eight non-overlapping peptides covering this 107 to 275 amino acid region were then synthesized and tested for their erythrocyte binding activity to further define the binding domains. Only two peptides, peptide P4 (at 171-191 amino acid position) and peptide P8 (at 255-275 amino acid position), were found to contain the erythrocyte binding activity. Competition assay revealed that each peptide recognizes its own erythrocyte receptor. These two peptides were found to be located on two parallel helices at one end of the protein in the modelled structure and could be exposed on its surface to form a suitable site for protein-protein interaction. Natural antibodies present in the sera of the P. vivax exposed individuals or the polyclonal rabbit antibodies against this protein were able to inhibit the erythrocyte binding activity of PvTRAg33.5, its fragments, and these two synthetic peptides P4 and P8. Further studies on receptor-ligand interaction might lead to the development of the therapeutic reagent.

BACKGROUND Tryptophan-rich antigens of malarial parasites have been proposed to be the potential vaccine candidate antigens. Plasmodium vivax contains the largest number of such antigens, which need to be evaluated for their immune responses. METHODS Recombinant proteins of 15 P. vivax tryptophan-rich antigens (PvTRAgs) were expressed, purified, and used for the human humoral and cellular immune responses. Genetic polymorphism of these 15 genes was also determined among clinical P. vivax isolates. RESULTS The T lymphocytes of P. vivax exposed individuals expressed higher level of CD69 against all 15 PvTRAgs. These antigens also activated the large population of CD4(+) T cells and produced higher level of intracellular IL-2, INF-γ and IL-4. Although there was a mixed Th1 and Th2 response against these antigens, this response was biased toward Th2. The majority of P. vivax patients (75.7%-100%, n = 33) produced IgG antibodies against these antigens. Most of these antigens showed conserved T- and B-cell epitopes in the parasite population. CONCLUSIONS These results suggest the presence of memory T cells in humans against these antigens to generate faster and more specific immune responses to minimize the P. vivax infection. Further characterization of these PvTRAgs may lead to the identification of a potential therapeutic target.

RTS,S is the most advanced malaria vaccine candidate, currently under phase-III clinical trials in Africa. This Plasmodium falciparum vaccine contains part of the central repeat region and the complete C-terminal T cell epitope region (Th2R and Th3R) of the circumsporozoite protein (CSP). Since naturally occurring polymorphisms at the vaccine candidate loci are critical determinants of the protective efficacy of the vaccines, it is imperative to investigate these polymorphisms in field isolates. In this study we have investigated the genetic diversity at the central repeat, C-terminal T cell epitope (Th2R and Th3R) and N-terminal T cell epitope regions of the CSP, in P. falciparum isolates from Madhya Pradesh state of India. These isolates were collected through a 5-year prospective study aimed to develop a well-characterized field-site for the future evaluation of malaria vaccine in India. Our results revealed that the central repeat (63 haplotypes, n = 161) and C-terminal Th2R/Th3R epitope (24 haplotypes, n = 179) regions were highly polymorphic, whereas N-terminal non-repeat region was less polymorphic (5 haplotypes, n = 161) in this population. We did not find any evidence of the role of positive natural selection in maintaining the genetic diversity at the Th2R/Th3R regions of CSP. Comparative analysis of the Th2R/Th3R sequences from this study to the global isolates (n = 1160) retrieved from the GenBank database revealed two important points. First, the majority of the sequences (similar to 61%, n = 179) from this study were identical to the Dd2/Indochina type, which is also the predominant Th2R/Th3R haplotype in Asia (similar to 59%, n = 974). Second, the Th2R/Th3R sequences in Asia, South America and Africa are geographically distinct with little allele sharing between continents. In conclusion, this study provides an insight on the existing polymorphisms in the CSP in a parasite population from India that could potentially influence the efficacy of RTS, S vaccine in this region.

Tryptophan-rich proteins from several malarial parasites have been identified where they play an important role in host-parasite interaction. Structural characterization of these proteins is needed to develop them as therapeutic targets. Here, we describe a novel Plasmodium vivax tryptophan-rich protein named PvTRAg33.5. It is expressed by blood stage(s) of the parasite and its gene contains two exons. The exon 1 encodes for a 23 amino acids long putative signal peptide which is likely to be cleaved off whereas the exon 2 encodes for the mature protein of 252 amino acids. The mature protein contains B-cell epitopes which were recognized by the human immune system during P.vivax infection. The PvTRAg33.5 contains 24 (9.5%) tryptophan residues and six motifs whose patterns were similar among tryptophan-rich proteins. The modeled structure of the PvTRAg33.5 consists of a multidomain architecture which is stabilized by the presence of large number of tryptophan residues. The recombinant PvTRAg33.5 showed predominantly α helical structure and alpha helix to beta sheet transition at pH below 4.5. Protein acquires an irreversible non-native state at temperature more than 50°C at neutral pH. Its secondary and tertiary structures remain stable in the presence of 35% alcohol but these structures are destabilized at higher alcohol concentrations due to the disturbance of hydrophobic interactions between tryptophanyl residues. These structural changes in the protein might occur during its translocation to interact with other proteins at its final destination for biological function such as erythrocyte invasion.