Non-collagenous Proteins Research Articles

Hierarchical Nature of Nanoscale Porosity in Bone Revealed by Positron Annihilation Lifetime Spectroscopy.

Bone is a hierarchical material primarily composed of collagen, water, and mineral that is organized into discrete molecular, nano-, micro-, and macroscale structural components. In contrast to the structural knowledge of the collagen and mineral domains, the nanoscale porosity of bone is poorly understood. In this study, we introduce a well-established pore characterization technique, positron annihilation lifetime spectroscopy (PALS), to probe the nanoscale size and distribution of each component domain by analyzing pore sizes inherent to hydrated bone together with pores generated by successive removal of water and then organic matrix (including collagen and noncollagenous proteins) from samples of cortical bovine femur. Combining the PALS results with simulated pore size distribution (PSD) results from collagen molecule and microfibril structure, we identify pores with diameter of 0.6 nm that suggest porosity within the collagen molecule regardless of the presence of mineral and water. We find that water occupies three larger domain size regions with nominal mean diameters of 1.1, 1.9, and 4.0 nm-spaces that are hypothesized to associate with intercollagen molecular spaces, terminal segments (d-spacing) within collagen microfibrils, and interface spacing between collagen and mineral structure, respectively. Subsequent removal of the organic matrix determines a structural pore size of 5-6 nm for deproteinized bone-suggesting the average spacing between mineral lamella. An independent method to deduce the average mineral spacing from specific surface area (SSA) measurements of the deproteinized sample is presented and compared with the PALS results. Together, the combined PALS and SSA results set a range on the mean mineral lamella thickness of 4-8 nm.

Spatial survey of non-collagenous proteins in mineralizing and non-mineralizing vertebrate tissues ex vivo

Bone biomineralization is a complex process in which type I collagen and associated non-collagenous proteins (NCPs), including glycoproteins and proteoglycans, interact closely with inorganic calcium and phosphate ions to control the precipitation of nanosized, non-stoichiometric hydroxyapatite (HAP, idealized stoichiometry Ca10(PO4)6(OH)2) within the organic matrix of a tissue. The ability of certain vertebrate tissues to mineralize is critically related to several aspects of their function. The goal of this study was to identify specific NCPs in mineralizing and non-mineralizing tissues of two animal models, rat and turkey, and to determine whether some NCPs are unique to each type of tissue. The tissues investigated were rat femur (mineralizing) and tail tendon (non-mineralizing) and turkey leg tendon (having both mineralizing and non-mineralizing regions in the same individual specimen). An experimental approach ex vivo was designed for this investigation by combining sequential protein extraction with comprehensive protein mapping using proteomics and Western blotting. The extraction method enabled separation of various NCPs based on their association with either the extracellular organic collagenous matrix phases or the inorganic mineral phases of the tissues. The proteomics work generated a complete picture of NCPs in different tissues and animal species. Subsequently, Western blotting provided validation for some of the proteomics findings. The survey then yielded generalized results relevant to various protein families, rather than only individual NCPs. This study focused primarily on the NCPs belonging to the small leucine-rich proteoglycan (SLRP) family and the small integrin-binding ligand N-linked glycoproteins (SIBLINGs). SLRPs were found to be associated only with the collagenous matrix, a result suggesting that they are mainly involved in structural matrix organization and not in mineralization. SIBLINGs as well as matrix Gla (γ-carboxyglutamate) protein were strictly localized within the inorganic mineral phase of mineralizing tissues, a finding suggesting that their roles are limited to mineralization. The results from this study indicated that osteocalcin was closely involved in mineralization but did not preclude possible additional roles as a hormone. This report provides for the first time a spatial survey and comparison of NCPs from mineralizing and non-mineralizing tissues ex vivo and defines the proteome of turkey leg tendons as a model for vertebrate mineralization.

Proteome Variation with Collagen Yield in Ancient Bone.

Isotope analyses are some of the most common analytical methods applied to ancient bone, aiding the interpretation of past diets and chronology. For this, the evaluation of “collagen yield” (as defined in radiocarbon dating and stable isotope research) is a routine step that allows for the selection of specimens that are deemed adequate for subsequent analyses, with samples containing less than ∼1% “collagen yield” normally being used for isotopic analysis but discounted for radiocarbon dating. The aims of this study were to use proteomic methods of MALDI–TOF (matrix assisted laser desorption ionization time-of-fligh mass spectrometry) and LC−ESI−MS/MS (liquid chromatography electrospray ionization tandem mass spectrometry) to investigate the endogeneity of the dominant proteinaceous biomolecules within samples that are typically considered to contain poorly preserved protein. Taking 29 archaeological samples, we evaluated the proteome variability between different acid-soluble fractions removed prior to protein gelatinization and considered waste as part of the radiocarbon dating process. We then correlated these proteomes against the commonly used “collagen yield” proxy for preservation. We found that these waste fractions contained a significant amount of both collagenous and noncollagenous proteins (NCPs) but that the abundance of these was not correlated with the acquired “collagen yield”. Rather than a depleted protein load as would be expected from a low “collagen yield”, the variety of the extracted NCPs was comparable with that commonly obtained from ancient samples and included informative proteins useful for species identification, phylogenetic studies, and potentially even for isotopic analyses, given further method developments. Additionally, we did not observe any correlation between “collagen yield” and peptide mass fingerprint success or between the different fractions taken from the same sample but at different radiocarbon pretreatment stages. Overall, these findings highlight the value in retaining and analyzing sample fractions that are otherwise discarded as waste during the radiocarbon dating process but more importantly, that low “collagen yield” specimens that are often misinterpreted by archaeologists as being devoid of protein can still yield useful molecular sequence-based information.

Biomineralization of Collagen-Based Materials for Hard Tissue Repair.

Hydroxyapatite (HA) reinforced collagen fibrils serve as the basic building blocks of natural bone and dentin. Mineralization of collagen fibrils play an essential role in ensuring the structural and mechanical functionalities of hard tissues such as bone and dentin. Biomineralization of collagen can be divided into intrafibrillar and extrafibrillar mineralization in terms of HA distribution relative to collagen fibrils. Intrafibrillar mineralization is termed when HA minerals are incorporated within the gap zone of collagen fibrils, while extrafibrillar mineralization refers to the minerals that are formed on the surface of collagen fibrils. However, the mechanisms resulting in these two types of mineralization still remain debatable. In this review, the evolution of both classical and non-classical biomineralization theories is summarized. Different intrafibrillar mineralization mechanisms, including polymer induced liquid precursor (PILP), capillary action, electrostatic attraction, size exclusion, Gibbs-Donnan equilibrium, and interfacial energy guided theories, are discussed. Exemplary strategies to induce biomimetic intrafibrillar mineralization using non-collagenous proteins (NCPs), polymer analogs, small molecules, and fluidic shear stress are discussed, and recent applications of mineralized collagen fibers for bone regeneration and dentin repair are included. Finally, conclusions are drawn on these proposed mechanisms, and the future trend of collagen-based materials for bone regeneration and tooth repair is speculated.

Reversible inhibition of trypsin activity with soybean flour in hide bating process for leather quality improvement

Trypsin is used to remove non-collagenous proteins from cattle hide in leather processing but prone to damage hide surface and reduce leather quality due to its rapid protein hydrolysis action on the hide surface and slow penetration through the whole hide. A novel trypsin bating method was developed using a reversible inhibition of trypsin with high-yield, low-cost and environment-friendly soybean flour (SF). When the mass ratio of trypsin to SF was 1:20, the trypsin activity was decreased by 77 % at a reaction time of 5 min and completely returned in 100 min because of the competitive Kunitz trypsin inhibitor in SF. Bating with trypsin-SF mixture avoided the damage to hide surface caused by trypsin, improved the dispersion uniformity of hide collagen fibers and increased the softness and the mechanical strength of leather. This work exhibits huge potential for improving the leather quality and provides new insights into the soybean application.

Evaluation of the effect of a remineralising paste containing strontium doped nanohydroxyapatite with Non-Collagenous protein analogue - chitosan on the characteristics of partially demineralized dentin -An invitro study

Preservation of tooth structure by remineralisation of affected dentin can contribute greatly to the concept of minimal invasive dentistry. Remineralisation of dentin is a challenge due to the absence of seeding crystals. Biomimetic remineralising approach aids in restoring demineralised dentin close to that of normal dentin with the use of non-collagenous protein (NCP) analogues that stabilize the mineral phase of dentin resulting in intrafibrillar remineralisation. This study was done to assess the effect of a remineralising paste containing strontium doped nanohydroxyapatite (Sr-nHAp) with non-collagenous protein (NCP) analogue chitosan on the characteristics of partially demineralised dentin. Experimental pastes were formulated using 10 & 20% Strontium doped nano-hydroxyapatite (nHAp) with or without NCP analogue-chitosan. After characterization by x-ray diffraction, its remineralising efficacy on partially demineralised dentin was assessed through SEM-EDS analysis and nanoindentation and compared to that of demineralised and normal dentin. It was observed that 20% Sr-nHAp paste and the experimental formulations with Sr-nHAp and NCP analogue chitosan had the potential to remineralise the partially demineralised dentin specimens as observed by both increase in the mineral content noted in the SEM-EDS analysis and increased mechanical properties analysed by nanoindentation. Within the limitations of the study it was observed that Chitosan enhanced remineralisation potential of 10 & 20% Sr-nHAP and improved mechanical properties of partially demineralised dentin and hence has potential application as a remineralising agent and in vital pulp therapy procedures.

Antimicrobial activity of a remineralizing paste containing Strontium doped Nano hydroxyapatite (Sr-nHAp) with Non Collagenous Protein (NCP) analogue Chitosan – An in vitro study

Dental caries is an infectious multifactorial disease. Biomimetic remineralisation of non-infected carious dentine helps in retaining tissues to a maximum degree during vital pulp treatment. However micro gaps and residual bacteria below restorations lead to microleakage, degradation of tooth structure and secondary caries. Remineralizing agent with anti-bacterial efficacy would help contain secondary caries and its sequelae. The aim of the study was to evaluate the antimicrobial activity of remineralizing paste containing Strontium doped Nano hydroxyapatite (Sr-nHAp) with Non Collagenous Protein (NCP) analogue, Chitosan. In this study dentin slabs, 200 µm thick obtained from freshly extracted premolars were demineralised and inoculated with Streptococcus mutans to simulate artificial carious lesion. The specimens were divided in to six groups based on the reminerlising regimen, Group 1–10 wt%Sr-nHAp, Group 2–20 wt%Sr-nHAp, Group 3–10 wt%Sr-nHAp + Chitosan and Group 4–20 wt%Sr-nHAp + Chitosan, Group 5 Positive control – Calcium hydroxide and Group 6, negative control, no treatment. The dentin specimens were homogenised and assessment of bactericidal effects was done by counting the number of viable bacteria after 48 h of incubation. Agar well diffusion test was performed to measure the zone of inhibition. Results were tabulated and analysed statistically using, ANOVA and Post – hoc Bonferroni tests (P = 0.05). All experimental groups except Group 1, exhibited antibacterial properties observed by Zone of Inhibition and reduced colony forming units. Antibacterial efficacy of the remineralizing paste was enhanced by the addition of Chitosan and its antimicrobial efficacy was comparable to calcium hydroxide.

ASPECTOS QUÍMICOS DA BIOMINERALIZAÇÃO ÓSSEA

THE CHEMISTRY BEHIND BONE BIOMINERALIZATION. The bone formation process is an example of how both time and nature acted harmonically to pave the path for life as we know. The unique association between calcium phosphate (CaP) and collagen in a single building block, gave rise to one of the most remarkable biomaterials that we know. Our aim in this revision is to provide a brief glance at the different organization levels of the bone tissue, by describing the main their main features. We start with a brief discussion regarding the thermodynamic conditions for mineral formation and how living organisms surpass them. Then we present collagen, the major organic component of bone, in the light of its hierarchical organization as well as its association to CaP crystals. The mechanisms that control both mineral formation and the association with the organic phase are further presented, in whichcells and other components of the extracellular matrix, such as matrix vesicles and noncollagenous proteins, become agents of bone construction. Finally, we underline the difficulty of studying the complex process of bone mineralization and present liposomes, proteoliposomes, and Langmuir monolayers as biomimetic approaches to this end.

The Structure of Bones Having or Lacking Osteocytes Exhibits Differences in Collagen Molecular Organization, Mineral Packing and the Organic-Inorganic Interfaces

Bone is a fascinating biomaterial comprised mostly of type-I collagen fibers as an organic phase, apatite as an inorganic phase, with water molecules residing at the interfaces between these phases. They are hierarchically organized with minor constituents such as non-collagenous proteins, citrate ions and glycosaminoglycans into a composite structure that is mechanically durable yet contains enough porosity to accommodate cells and blood vessels. The nanometer scale organization of the collagen fibrous structure and the mineral constituents in bone were recently extensively scrutinized. However, molecular details at the lowest hierarchical level still need to be unraveled to better understand the exact atomic-level arrangement of all these important components in the context of the integral structure of the bone.

Polyelectrolyte-calcium complexes as a pre-precursor induce biomimetic mineralization of collagen.

Polyelectrolytes such as polyaspartic acid (PAsp) are critical in biomimetic mineralization as stabilizers of amorphous calcium phosphate (ACP) precursors and as nucleation inhibitors similar to non-collagenous proteins (NCPs). Nevertheless, the application of polyelectrolyte-calcium complexes as a pre-precursor, such as PAsp-Ca complexes, in the mineralization of collagen is unexplored. Herein, we propose a polyelectrolyte-Ca complex pre-precursor (PCCP) process for collagen mineralization. By combining three-dimensional (3D) STORM, potential measurements, and cryogenic transmission electron microscopy with molecular dynamics simulations, we show that liquid-like electropositive PAsp-Ca complexes along with free calcium ions infiltrate electronegative collagen fibrils. The PAsp-Ca complexes are immobilized within the fibrils via chelation and hydrogen bonds, and outward movement of free calcium ions is prevented while phosphate and hydroxide are recruited through electrostatic attractions. Afterwards, ACP instantly forms and gradually crystallizes. The PCCP process not only unites two distinct crystallization pathways (classical (free Ca/P ions) and non-classical (polyelectrolyte-Ca complexes)), but also provides a novel strategy for rapid biomimetic mineralization of collagen.

Effect of nano-hydroxyapatite with biomimetic analogues on the characteristics of partially demineralised dentin: An in-vitro study.

Research on dentin remineralisation protocols in particular 'biomimetic remineralisation' has gained huge momentum. Aim of this study was to evaluate if biomimetic analogs, incorporated in n-HAp, as an experimental formulation could aid in remineralization of artificial caries-like dentin and have anti-microbial effect on cariogenic bacteria, S mutans. An experimental paste was formulated using nano-hydroxyapatite (nHAp) with Non-Collagenous Protein analogs- polyacrylic acid (PAA), sodium tri-poly phosphate (STPP) with Simulated Body Fluid. Partially demineralised dentin specimens were divided into three groups (n=10) based on the remineralisation treatment as, Group A- n-HAp paste, Group B- n-HAp and NCP analogues and Group C (Control) - no treatment. At the end of the experimental period, the specimens were assessed using SEM-EDS analysis and Vickers microhardness testing. Further, the antimicrobial efficacy of the paste was assessed. The results were statistically analyzed using ANOVA with post-hoc Bonferroni test. Dentin specimens treated with the experimental paste revealed greater tubular occlusion, with intra tubular deposits and increased mineral content. Specimens treated with n-HAp alone had higher microhardness values and inhibitory effect on the cariogenic bacteria. Non-Collagenous Protein analogs incorporated in n-HAp could remineralize the demineralised dentin and had antibacterial efficacy against S mutans.

Editorial introductions

Editorial introductions

Dual function of poly(acrylic acid) on controlling amorphous mediated hydroxyapatite crystallization

Hydroxyapatite (HAP, Ca5(PO4)3OH), as the main inorganic component of bone, is regulated by noncollagenous proteins (NCPs) that are rich in acidic amino acid. Poly(acrylic acid) (pAA) is widely used as an NCPs mimic because of its rich in carboxyl acid groups. Here we show that pAA plays a dual role in amorphous calcium phosphate (ACP, Ca3(PO4)2) mediated HAP crystallization, in which ACP is usually considered as the precursor phase of HAP crystallization in vitro and in vivo. The appropriate concentration of pAA can promote HAP crystallization due to it is mainly incorporated into ACP and more likely leads to form smaller ACP nanoparticles, thus provide a large specific surface area to HAP nucleation. While excessive pAA (incorporated into and adsorbed on the ACP) impairs the crystallization of HAP, in that surface associated pAA prevent the nucleation of HAP on ACP. Interestingly, pAA adsorbed on the ACP surface inhibits HAP nucleation significantly and changes the aggregation events of ACP. This finding points out that appropriate regulation of pAA concentration is vital for amorphous mediated biomineralization. Moreover, this study is of considerable importance for the future regulation and design of bone substitutes associated with NCPs.

Deciphering the Relevance of Bone ECM Signaling.

Bone mineral density, a bone matrix parameter frequently used to predict fracture risk, is not the only one to affect bone fragility. Other factors, including the extracellular matrix (ECM) composition and microarchitecture, are of paramount relevance in this process. The bone ECM is a noncellular three-dimensional structure secreted by cells into the extracellular space, which comprises inorganic and organic compounds. The main inorganic components of the ECM are calcium-deficient apatite and trace elements, while the organic ECM consists of collagen type I and noncollagenous proteins. Bone ECM dynamically interacts with osteoblasts and osteoclasts to regulate the formation of new bone during regeneration. Thus, the composition and structure of inorganic and organic bone matrix may directly affect bone quality. Moreover, proteins that compose ECM, beyond their structural role have other crucial biological functions, thanks to their ability to bind multiple interacting partners like other ECM proteins, growth factors, signal receptors and adhesion molecules. Thus, ECM proteins provide a complex network of biochemical and physiological signals. Herein, we summarize different ECM factors that are essential to bone strength besides, discussing how these parameters are altered in pathological conditions related with bone fragility.

Effect of aspartic acid on the crystallization kinetics of ACP and dentin remineralization

Type I collagen and non-collagen proteins are the main organic components of dentin. This study aimed to investigate the biomimetic remineralization of demineralized dentin by aspartic acid (Asp), which is abundant in non-collagenous proteins (NCPs). Asp was added to a mineralizing solution containing polyacrylic acid (PAA) to explore the mechanism of Asp regulating the pure amorphous calcium phosphate (ACP) phase transition process. The remineralization process and superstructure of the remineralized layer of demineralized dentin were evaluated and analyzed by transmission electron microscope (TEM) and scanning electron microscope (SEM), and the biological stability of the remineralized layer was investigated by collagenase degradation experiment. It demonstrated that Asp promoted the crystallization kinetics of PAA-stabilized amorphous calcium phosphate to hydroxyapatite (HAP), and shortened the remineralization time of demineralized dentin from 7 days to 2 days. The newly formed remineralized dentin had similar morphology and biological stability to the natural dentin layer. The presence of a large number of Asp residues in NCPs promoted the phase transformation of ACP, and further revealed the mechanism of action of NCPs in dentin biomineralization. This experiment also showed that Asp promoted the biomimetic remineralization of dentin; the morphology and hierarchical structure of remineralized layer was similar to that of natural teeth, and had good biological properties.

ProAlanase is an Effective Alternative to Trypsin for Proteomics Applications and Disulfide Bond Mapping

Trypsin is the protease of choice in bottom-up proteomics. However, its application can be limited by the amino acid composition of target proteins and the pH of the digestion solution. In this study we characterize ProAlanase, a protease from the fungus Aspergillus niger that cleaves primarily on the C-terminal side of proline and alanine residues. ProAlanase achieves high proteolytic activity and specificity when digestion is carried out at acidic pH (1.5) for relatively short (2 h) time periods. To elucidate the potential of ProAlanase in proteomics applications, we conducted a series of investigations comprising comparative multi-enzymatic profiling of a human cell line proteome, histone PTM analysis, ancient bone protein identification, phosphosite mapping and de novo sequencing of a proline-rich protein and disulfide bond mapping in mAb. The results demonstrate that ProAlanase is highly suitable for proteomics analysis of the arginine- and lysine-rich histones, enabling high sequence coverage of multiple histone family members. It also facilitates an efficient digestion of bone collagen thanks to the cleavage at the C terminus of hydroxyproline which is highly prevalent in collagen. This allows to identify complementary proteins in ProAlanase- and trypsin-digested ancient bone samples, as well as to increase sequence coverage of noncollagenous proteins. Moreover, digestion with ProAlanase improves protein sequence coverage and phosphosite localization for the proline-rich protein Notch3 intracellular domain (N3ICD). Furthermore, we achieve a nearly complete coverage of N3ICD protein by de novo sequencing using the combination of ProAlanase and tryptic peptides. Finally, we demonstrate that ProAlanase is efficient in disulfide bond mapping, showing high coverage of disulfide-containing regions in a nonreduced mAb.

Improving the colonization and functions of Wharton’s Jelly-derived mesenchymal stem cells by a synergetic combination of porous polyurethane scaffold with an albumin-derived hydrogel

The development of neo-tissues assisted by artificial scaffolds is continually progressing, but the reproduction of the extracellular environment surrounding cells is quite complex. While synthetic scaffolds can support cell growth, they lack biochemical cues that can prompt cell proliferation or differentiation. In this study, Wharton’s Jelly-derived mesenchymal stem cells are seeded on a polyurethane (PU) scaffold combined with a hydrogel based on bovine serum albumin (BSA). BSA hydrogel is obtained through thermal treatment. While such treatment leads to partial unfolding of the protein, we show that the extent of denaturation is small enough to maintain its bioactivity, such as protein binding. Therefore, BSA provides a suitable playground for cells inside the scaffold, allowing higher spreading, proliferation and matrix secretions. Furthermore, the poor mechanical properties of the hydrogel are compensated for by the porous PU scaffold, whose architecture is well controlled. We show that even though PU by itself can allow cell adhesion and protein secretion, cell proliferation is 3.5 times higher in the PU + BSA scaffolds as compared to pure PU after 21 d, along with the non-collagenous protein secretions (389 versus 134 μmmg−1). Conversely, the secretion of sulphated glycosaminoglycans is 12.3-fold higher in the scaffold made solely of PU. Thereby, we propose a simple approach to generating a hybrid material composed of a combination of PU and BSA hydrogel as a promising scaffold for tissue regeneration.

Morphological Characterization of Deciduous Enamel and Dentin in Patients Affected by Osteogenesis Imperfecta

The purpose of this study was to clarify the structural and ultrastructural alterations of the enamel and dentin collagen network in the deciduous teeth of children affected by osteogenesis imperfecta (OI) using field-emission in-lens scanning electron microscopy (FEI-SEM) and transmission electron microscopy (TEM) analyses. Exfoliated primary teeth were collected from children with a diagnosis of OI and from healthy individuals (N = 24). Tooth slices containing both dentin and enamel were fixed, dehydrated and dried, gold sputtered, and observed using FEI-SEM. Additional dentin fragments were decalcified, dehydrated, embedded in resin, cut, and processed for TEM analysis. Under FEI-SEM, the enamel in OI-affected children showed an irregular prism distribution with the enamel hydroxyapatite crystals unpacked. Ultrastructural correlative analysis of the dentin in patients affected by OI showed an altered collagen pattern with a low density. In some areas, teeth in OI patients showed a reduction in the number of dentinal tubules, with odontoblastic process missing in most of the tubules. The presence of altered dentine and enamel organization in OI children was firmly established at an ultrastructural level, but additional biochemical studies are necessary in order to clarify quantitatively and qualitatively the collagenic and non-collagenic proteins in this disorder.

Rescuing key native traits in cultured dermal papilla cells for human hair regeneration

IntroductionThe dermal papilla (DP) represents the major regulatory entity within the hair follicle (HF), inducing hair formation and growth through reciprocal interactions with epithelial cells. However, human DP cells rapidly lose their hair inductive ability when cultured in an epithelium-deficient environment. ObjectivesTo determine if the conditioned medium collected from interfollicular keratinocytes (KCs-CM) is capable of improving DP cell native properties and inductive phenotype. MethodsDP cells were cultured with KCs-CM both in 2D and 3D culture conditions (spheroids). Further, the hair-inductive capacity of DP cells precultured with KCs-CM was tested in a hair reconstitution assay, after co-grafting with human keratinocytes in nude mice. ResultsWe demonstrate that KCs-CM contributes to restore the inductivity of cultured human DP cells in a more effective mode than the conventional 3D-cultures. This is supported by the higher active alkaline phosphatase (ALP) levels in DP cells, the improved self-aggregative capacity and the reduced expression of α-SMA and the V1-isoform of versican. Moreover, DP cells cultured with KCs-CM displayed a secretome profile (VEGF, BMP2, TGF- β1, IL-6) that matches the one observed during anagen. KCs-CM also enhanced DP cell proliferation, while preventing cells to undergo morphological changes characteristic of high passage cells. In opposition, the amount of collagenous and non-collagenous proteins deposited by DP cells was lower in the presence of KCs-CM. The improvement in ALP activity was maintained in 3D spheroidal cultures, even after KCs-CM retrieval, being superior to the effect of the gold-standard culture conditions. Moreover, DP cells cultured with KCs-CM and grafted with human keratinocytes supported the formation of HF- and sebaceous gland-like structures in mice. ConclusionThe proposed strategy encourages future cell-based strategies for HF regeneration not only in the context of hair-associated disorders, but also in the management of wounds to aid in restoring critical skin regulatory appendages.

Single-Molecule Force Spectroscopy Reveals Adhesion-by-Demand in Statherin at the Protein-Hydroxyapatite Interface.

Achieving strong adhesion in wet environments remains a technological challenge in biomedical applications demanding biocompatibility. Attention for adhesive motifs meeting such demands has largely been focused on marine organisms. However, bioadhesion to inorganic surfaces is also present in the human body, in the hard tissues of teeth and bones, and is mediated through serines (S). The specific amino acid sequence DpSpSEEKC has been previously suggested to be responsible for the strong binding abilities of the protein statherin to hydroxyapatite, where pS denotes phosphorylated serine. Notably, similar sequences are present in the non-collagenous bone protein osteopontin (OPN) and the mussel foot protein 5 (Mefp5). OPN has previously been shown to promote fracture toughness and physiological damage formation. Here, we investigated the adhesion strength of the motif D(pS)(pS)EEKC on substrates of hydroxyapatite, TiO2, and mica using atomic force microscopy (AFM) single-molecule force spectroscopy (SMFS). Specifically, we investigated the dependence of adhesion force on phosphorylation of serines by comparing findings with the unphosphorylated variant DSSEEKC. Our results show that high adhesion forces of over 1 nN on hydroxyapatite and on TiO2 are only present for the phosphorylated variant D(pS)(pS)EEKC. This warrants further exploitation of this motif or similar residues in technological applications. Further, the dependence of adhesion force on phosphorylation suggests that biological systems potentially employ an adhesion-by-demand mechanism via expression of enzymes that up- or down-regulate phosphorylation, to increase or decrease adhesion forces, respectively.