The Atlantic cod (Gadus morhua) and red king crab (Paralithodes camtschaticus) processing wastes are massive and unutilized in the Murmansk region of Russia. The samples of skin-containing waste of Atlantic cod fillets production were hydrolyzed using enzyme preparations derived from red king crab hepatopancreases, porcine pancreases and Bacillus subtilis bacteria. The activity of enzymes from crab hepatopancreases was significantly higher than the activity of enzymes derived from other sources. The optimal conditions of the hydrolysis process have been figured out. The samples of cod processing waste hydrolysate were analyzed for amino acid composition and molecular weight distribution. The samples of hydrolysate were used as core components for bacterial culture medium samples. The efficiency of the medium samples was tested for Escherichia coli growth rate; the most efficient sample had efficiency 95.3% of that of a commercially available medium based on fish meal. Substitution of medium components with those derived from industrial by-products is one of the ways to decrease a cost of a culture medium in biopharmaceutical drug production.

More than 800 million tons of fish are utilizing in a year and 25-30% become waste. The waste amount is beneficial source for extraction of collagen. But procedure of extraction is still to be optimized. The current study was designed to extract collagen from fish through fish waste fermentation under various conditions. For collagen extraction we did lactic acid fermentation in which yogurt and Dough bacteria was added with fish sample and placed it in incubator at 30ºC for one month. In yogurt and dough culture have at least 10 type of lactic acid bacterial species. After every week we check PH of each sample and take soup of that sample. After centrifugation of that sample’s TCA (Trichloroacetic acid) precipitation was done of yogurt and dough sample and kept at -20ºC. then did SDS-PAGE) using 6% resolving and 5% stacking gel of already stored sample. Then HaCaT cells (1 × 10⁴ cells/well) were cultured in 96-well flat-bottom culture plates and treated with appropriate doses of FFCP (fermented fish collagen peptide) for 24 and 48 hours.The SDS-PAGE revealed that the collagen protein of fish had doublet pattern for α1 and α2 chains at corresponding to 145 kDa and 132 kDa respectively. The density for α1 twice as compared to α2. Our result agrees that The fish collagen consists mostly of α-chain as well as little amount of inter and intra molecular cross-linked components of α-chains; b (dimmer) and c (trimer). Different biochemical tests were done for identification of lactic acid bacteria catalase positive, citrate positive, urease negative. Our fermented fish collagen and peptides mixture were test for cellular cytotoxicity and proliferative effect on HaCaT cells. Current result shows that fermented extracted collagen are nontoxic and induce the proliferation of HaCaT cells. Current study is supported by various studies that revealed the medical application of fish extracted collagen as underline.

In this study, an extracellular metalloprotease from Pseudoalteromonas sp. H2 was purified and identified. The EH2 maintained more than 80% activity over a wide pH range of 5-10 and the stability was also nearly independent of pH. More than 65% of activity was detected in a wide temperature of 20-70℃. The high stability of the protease in presence of different surfactants and oxidizing agent was also observed. Moreover, we also investigated the antioxidant activities of the hydrolysates generated from porcine and salmon skins collagen by EH2. The results show that salmon skins collagen hydrolysates demonstrated higher DPPH (42.88%±1.85) and hydroxyl radical (61.83%±3.05) scavenging activity compared to those produced with porcine skins collagen. For oxygen radical absorbance capacity, the hydrolysates from porcine skins collagen had higher efficiency (7.72±0.13 μmol·TE/μmol). Even 1 nM mixed peptides can effectively reduce the levels of intracellular ROS. And the two type of substrates exerted the best antioxidant activity when hydrolyzed for 3 hours. Hydrolysis time and type of substrate exerted important effect on the antioxidant property of hydrolysates. This study may have implications for the potential application of marine protease in biocatalysis industry. In addition, the hydrolyzed peptides from meat waste containing beneficial collagen by protease have good antioxidant activity indicating it may be a potential additive in the food processing industry and cosmetics industry.

There is a growing need for novel in vitro corneal models to replace animal-based ex vivo test in drug permeability studies. In this study we demonstrate a corneal mimetic that models the stromal and epithelial compartments of human cornea. Human corneal epithelial cells (HCE-T) were grown on top of a self-supporting porcine collagen-based hydrogel. Cross sections of the multilayers were characterized by histological staining and immunocytochemistry of zonula occludens-1 protein (ZO-1) and occludin. Furthermore, water content and elastic properties of the synthetized collagen type I-based hydrogels were measured. The apparent permeability coefficient (Papp) values of a representative set of ophthalmic drugs were measured and correlated to rabbit cornea Papp values found in the literature. Multilayered structure of HCE-T cells and expression of ZO-1 and occludin in full thickness of multilayer were observed. The hydrogel-based corneal model exhibited excellent correlation to rabbit corneal permeability (r=0.96), whereas insert-grown HCE-T multilayer was more permeable and the correlation to the rabbit corneal permeability was lower (r=0.89). The hydrogel-based human corneal model predicts the rabbit corneal permeability more reliably in comparison to HCE-T cells grown in inserts. This in vitro human corneal model can be successfully employed for drug permeability tests whilst avoiding ethical issues and reducing costs.

Major progress in the field of regenerative medicine are expected from the design of artificial scaffolds that mimic both the structural and functional properties of the ECM. The bionanocomposites approach is particularly well fitted to meet this challenge as it can combine ECM-based matrices and colloidal carriers of biological cues that regulate cell behavior. Here we have prepared bionanocomposites under high magnetic field from Tilapia fish scale collagen and multifunctional silica nanoparticles (SiNPs). We show that scaffolding cues (collagen), multiple display of signaling peptides (SiNPs) and control over the global structuration (magnetic field) can be combined into a unique bionanocomposite for the engineering of biomaterials with improved cell performances.

Skin aging is one of the most common problems facing humanity. It occurs because of altering the balance between free radicals and antioxidants and increasing the amount of the reactive oxygen species (ROS) in skin cells, which leads to oxidative stress especially when exposed to UV radiation. Antioxidants can neutralize the harmful effects of ROS, and secondary plant metabolites can help protect against UV radiation. In this study, punicalagin was extracted from pomegranate and concentrations of total polyphenolics and flavonoids were determined and antioxidant activities measured. Punicalagin was loaded onto niosomes and its morphology and release were studied. An in vitro study was performed on human fibroblast cell line HFB4 cells with aging induced by H2O2 and UV radiation. Cell cycle arrest was studied and different genes (MMP3, Col1A1, Timp3, and TERT) involved in the skin aging process were selected to measure punicalagin's effect. Punicalagin succeeded in reducing the growth arrest of HFB4 cells, activated production of the Col1A1 and Timp3 genes, maintained collagen level, and lowered of MMP3. Punicalagin increased human TERT concentration in skin cells. Punicalagin is promising as a natural antioxidant to protect human skin from aging.

Mandibular critical size defect (CSD) due to pathological conditions, trauma, and congenital disease can not heal spontaneously and predominantly filled with fibrous tissue. Therefore, a Guided Bone Regeneration (GBR) combined with bone grafting can be performed. The researchers considered using Demineralized Dentin Material Membrane (DDMM) from bovine dentine as an alternative GBR. This study aimed to determine the amount of fibroblast and collagen density after DDMM and bone graft implantation on CSD. Thirty-six Rattus norvegicus rats were used as samples. Mandibular bone defect 5x5 mm was made, then filled with bone graft and covered with Bovine Pericardium Collagen Membrane (BPCM) in the control group and DDMM in the treatment group. Six samples were sacrificed on 7, 14, and 21 days post-surgical for histology examination. There were no significant differences in the amount of fibroblast and collagen density (p-value > 0,05). The amount of fibroblast is lower and the collagen density is higher in treatment group. DDMM has microporosity to prevent connective tissue ingrowth and dentine tubules to allow growth factors release. DDMM and bone graft implantation can reduce the amount of fibroblasts and increase collagen density of CSD which potentially being used as a CSD alternative treatment for bone regeneration.

We set a feasible method to produce tailored collagen scaffolds and analyzed its potential for corneal engineering. Collagen-vitrigel membranes (CVM) were produced with a 1:1 ratio of Dulbecco’s Modified Eagle’s medium (DMEM), 1% antibiotics and 8% fetal bovine serum, and 5mg/mL collagen type I. Three volumes of collagen were used: 1X (2.8 L/mm2 of collagen), 2X, and 3X. Vitrification was done at 40% relative humidity (RH), 40° C, and 30 rpm using a matryoshka system set with a shaking-oven and a desiccator with a saturated K2CO3 solution. The CVM was characterized for width, microstructure, transparency, and biocompatibility using NIH3T3 cells. Surgical manipulation was assessed in an ex vivo corneal model. Constructs of corneal endothelial cells (CECs) and 2X-CVM were transplanted into five 18-month-old White New Zealand rabbits. CVM exhibited homogeneous surface and laminar organization. Membrane width increased with gel volume from 3.65µm to 7.2µm. 1X and 2X-CVM exhibited a 99% transmittance. NIH3T3 cells concentration increased 3-fold within 48 h with no significant difference among the 3 CVM (p = 0.323). The 2X-CVM was surgically manipulable. Transplantation of corneal endothelial cells (CECs) seeded over 2X-CVM restored corneal endothelium. The matrioshka system is a feasible method that yields CVM suitable for corneal engineering.

Abstract: The effect of collagen type 1 (Col I) oxidation on Adipose Tissue-Derived Mesenchymal Stem Cells (ADMSCs) remodeling is described as a model for acute oxidative stress. Morphologically, remodeling was presented by a mechanical rearrangement of adsorbed FITC-Col I and a trend for its organization in a fibril-like pattern - a process strongly abrogated in oxidized samples, but without visible changes in cell morphology. The cellular proteolytic activity was quantified in multiple samples utilizing fluorescence de-quenching (FRET effect). In the presence of ADMSCs a significant increase of native FITC-Col I fluorescence was observed, almost absent in the oxidized samples. Parallel studies in cell-free systems confirmed the enzymatic de-quenching of native FITC-Col I by Clostridial collagenase, again showing significant inhibition in oxidized samples. The structural changes in the oxidized Col I was further studied by Differential Scanning Calorimetry: an additional endotherm at 33.6°C along with the typical for native Col I at 40.5°C with sustained enthalpy (∆H) was observed in oxidized samples. Collectively, it has been evidenced that remodeling of Col I by ADMSCs is altered upon oxidation due to the intrinsic changes in the protein structure, thus presenting a novel mechanism for the control of stem cells' behavior toward collagen.

Previous study demonstrated the reconstituted type I collagen matrix extracted from rabbit tendons enabled to regenerate the TMJ disc in the rabbit. The aim of this study was to investigate changes in the extracellular matrix (ECM) and mechanisms of regeneration in TMJ disc. In 36 New Zealand rabbits that underwent a partial discectomy, discs were replaced with reconstituted collagen templates for 3 months. A histological analysis showed that moderate to severe degeneration appeared in partially discectomized joints without implantation. In contrast, discs that received the reconstituted collagen template regenerated, and returned to normal to protect the joint. Cells in the regenerative tissue expressed ECM, and fibers became regular and compact due to tissue remodeling over time. Reparative cells differentiated into chondroblasts, and showed highly dense pericellular fibers. The morphology and collagen composition of the disc and condyle in the 3-month experimental group were similar to those of normal tissues. In conclusion, the reconstituted collagen template facilitated the regeneration of surgically discectomized discs. Type I and type II collagens play a crucial role in the regeneration of articular discs.

The synthesis of new collagen, chitosan and chitosan-collagen coated magnetic nanoparticles have been done. Two types of cross-linkers for polymer shell stabilization were used: glutaraldehyde (Gla) as a standard cross-linker and new one – squaric acid (SqA). Structure and morphology of prepared nanoparticles were characterized by ATR-FT IR, XRD and TEM analysis. The immobilization of lipase from Candida rugosa was performed on the nanoparticles surface. The amount of immobilized enzyme was quantified by the Bradford method. All of lipase-biopolymers coated nanoparticles were characterized with good activity recovery. A little hyperactivation of lipase immobilized on nanoparticles with SqA was observed. All of prepared lipase-immobilized nanoparticles were characterized with very good reusability.

Severe respiratory syncytial virus (RSV) infections in early life have been linked to the development of chronic airway disease. RSV is a potent inducer of reactive oxygen species (ROS) which contributes to inflammation and enhanced clinical disease. NF-E2-related factor 2 (Nrf2) is an evolutionary conserved redox-responsive protein that helps to protect cells and whole organisms from oxidative stress and injury. The role of Nrf2 in the context of viral-mediated chronic lung injury is not known. Herein, we show that RSV experimental infection of adult Nrf2 deficient BALB/c mice (Nrf2-/-; Nrf2 KO) is characterized by enhanced disease, increased inflammatory cell recruitment to the bronchoalveolar compartment, and a more robust upregulation of innate and inflammatory genes and proteins, compared to wild type Nrf2+/+ competent mice (WT). These events that occur at very early time points, lead to increased peak RSV replication in Nrf2 KO compared to WT mice (day 5). To evaluate longitudinal changes of the lung architecture, mice were scanned weekly by high-resolution micro-computed tomography (micro-CT) imaging up to 28 days after initial viral inoculation. Based on micro-CT qualitative 2D imaging and quantitative reconstructed histogram-based analysis of lung volume and density, we found that RSV infected Nrf2 KO mice developed significantly greater and prolonged fibrosis compared to WT mice. Results of this study underscore the critical role of Nrf2-mediated protection from oxidative injury not only in acute pathogenesis of RSV infection, but also in its long-term consequences of chronic airway injury.

Mesenchymal stromal cells (MSCs) have shown a high potential for cartilage repair. Collagen-based scaffolds are used to deliver and retain cells at the site of cartilage damage. The aim of the work was a comparative analysis of the capacity of the MSCs from human adipose tissue to differentiate into chondrocytes in vitro and to stimulate the regeneration of articular cartilage in an experimental model of rabbit knee osteoarthrosis when cultured on microheterogenic collagen-based hydrogel (МCH) and the microparticles of decellularized porcine articular cartilage (DPC). The morphology of samples was evaluated using scanning electron microscopy and histological staining methods. On the surface of the DPC, the cells were distributed more uniformly than on the MCH surface. On day 28, the cells cultured on the DPC produced glycosaminoglycans more intensely compared to the MCH with the synthesis of collagen type II. However, in the experimental model of osteoarthrosis, the stimulation of the cartilage regeneration was more effective when the MSCs were administered to the MCH carrier. The present study demonstrates the way to regulate the action of the MSCs in the area of cartilage regeneration: the MCH is more conducive to stimulating cartilage repair by the MSCs, while the DPC is an inducer for a formation of a cartilage-like tissue by the MSCs in vitro.

Prediction of tumor consistency is valuable for planning transsphenoidal surgery for pituitary adenoma. A prospective study was conducted involving 49 participants with pituitary adenoma to determine whether quantitative pharmacokinetic analysis of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) is useful for predicting consistency of adenoma. Pharmacokinetic parameters in the adenomas including volume of extravascular extracellular space (EES) per unit volume of tissue (ve), blood plasma volume per unit volume of tissue (vp), volume transfer constant between blood plasma and EES (Ktrans), and rate constant between EES and blood plasma (kep) were obtained. The pharmacokinetic parameters and the histologic percentage of collagen content (PCC) were compared between soft and hard adenomas using Mann–Whitney U test. Pearson’s correla-tion coefficient was used to correlate pharmacokinetic parameters with PCC. Hard adenomas showed significantly higher PCC (44.08 ± 15.14% vs. 6.62 ± 3.47%, p < 0.01), ve (0.332 ± 0.124% vs. 0.221 ± 0.104%, p = 0.02), and Ktrans (0.775 ± 0.401/min vs. 0.601 ± 0.612/min, p = 0.02) than soft adenomas. Moreover, a significant positive correlation was found between ve and PCC (r = 0.601, p < 0.01). The ve derived using DCE-MRI may have predictive value for consistency of pituitary adenoma.

Accelerated degradation of collagen membranes (CMs) in diabetic rats is associated with increased infiltration of macrophages and blood vessels. Since pre-implantation immersion of CMs in cross-linked high molecular weight hyaluronic acid (CLHA) delays membrane degradation, we evaluated its effect on the number of macrophages and endothelial cells (ECs) within the CM. Diabetes was induced with streptozotocin in 16 rats, while 16 healthy rats served as control. CM discs were labeled with biotin, soaked in CLHA or PBS and implanted under the scalp. Fourteen days later, CMs were embedded in paraffin and the number of macrophages and ECs within the CMs was determined using antibodies against CD68 and Transglutaminase II, respectively. Diabetes increased the number of macrophages and ECs within the CMs (∼2.5-fold and 4-fold, respectively). Immersion of CMs in CLHA statistically significantly reduced the number of macrophages (p<0.0001) in diabetic rats, but not that of ECs. In the healthy group, CLHA had no significant effect on the number of either cells. Higher residual collagen area and membrane thickness in CLHA-treated CMs in diabetic animals were significantly correlated with reduced number of macrophages but not ECs. Immersion of CM in CLHA inhibits macrophage infiltration and reduces CM degradation in diabetic animals.

Rats fed a high-fat diet with a single streptozotocin (STZ) injection developed obesity, prediabetes, cardiac hypertrophy and diastolic dysfunction. Here we aimed to explore the renal consequences of prediabetes in the same groups of rats. Male Long-Evans rats were fed normal chow (CON; n = 9) or high-fat diet containing 40% lard and were administered STZ at 20 mg/kg (i.p.) at week four (prediabetic rats, PRED, n = 9). At week 21 cardiac functions were examined (Koncsos et al., 2016) and blood and urine samples were taken. Kidney samples were collected for histology, immunohistochemistry and for analysis of gene expression. High-fat diet and streptozotocin increased body weight gain and visceral adiposity, and plasma leptin, elevated fasting blood glucose levels, impaired glucose and insulin tolerance, despite hyperleptinemia, plasma C-reactive protein concentration decreased in PRED rats. Immunohistochemistry revealed elevated collagen IV protein expression in the glomeruli, and Lcn2 mRNA expression increased, while Il-1β mRNA expression decreased in both the renal cortex and medulla in PRED vs. CON rats. Kidney histology, urinary protein excretion, plasma creatinine, glomerular Feret diameter, desmin protein expression and cortical and medullary mRNA expression of TGF-β1, Nrf2, PPARγ were similar in CON and PRED rats. Reduced AMPKα phosphorylation of the autophagy regulator Akt was the first sign of liver damage, while serum lipid and liver enzyme levels were similar. In conclusion, glomerular collagen deposition and increased lipocalin-2 expression were the early signs of kidney injury, while most biomarkers of inflammation, oxidative stress and fibrosis were negative in the kidneys of obese, prediabetic rats with mild heart and liver injury.

Collagen, the most abundant structural protein found in mammals, plays a vital role as a constituent of the extracellular matrix (ECM) that surrounds cells. Collagen fibrils are strengthened through the formation of covalent cross-links, which involve complex enzymatic and non-enzymatic reactions. Lysyl oxidase (LOX) is responsible for catalyzing the oxidative deamination of lysine and hydroxylysine residues, resulting in the production of aldehydes, allysine, and hydroxyallysine. These intermediates undergo spontaneous condensation reactions, leading to the formation of immature cross-links, which are the initial step in the development of mature covalent cross-links. Additionally, non-enzymatic glycation contributes to the formation of abnormal cross-linking in collagen fibrils. During glycation, specific lysine and arginine residues in collagen are modified by reducing sugars, leading to the creation of Advanced Glycation End-products (AGEs). These AGEs have been associated with changes in the mechanical properties of collagen fibers. Interestingly, various studies have reported that plant polyphenols possess amine oxidase-like activity and can act as potent inhibitors of protein glycation. This review article focuses on compiling literature describing polyphenols with amine oxidase-like activity and antiglycation properties. Specifically, we explore the molecular mechanisms by which specific flavonoids impact or protect the normal collagen cross-linking process. Furthermore, we discuss how these dual activities can be harnessed to generate properly cross-linked collagen molecules, thereby promoting the stabilization of highly organized collagen fibrils.

A recently identified broiler myopathy known as wooden breast (WB) is predominantly found in the pectoralis major muscle of fast-growing broiler hybrids and is causing significant losses to the poultry industry. The aim of this study was to investigate the effects of WB syndrome on raw meat texture, purge loss and thermal properties of intramuscular connective tissue of pectoralis major muscle in the early post-mortem period. Results showed that WB muscles at 1d postmortem was associated with significantly increased stiffness and significantly increased purge loss compared to normal muscles (NB). However, at 3d postmortem, these parameters did not differ between the two groups. Insoluble and total collagen content was significantly higher in WB muscles compared to NB muscles, and the extractability of intramuscular connective tissue (IMCT) was also higher and remained stable in the early postmortem period. These were coincided with significantly lower protein content in the sarcoplasmic protein fraction and myofibrillar protein fraction of WB muscles compared to NB muscles (P < 0.05). The IMCT of these two groups showed different thermal properties, as the enthalpy of denaturation (ΔH) was significantly lower in WB muscles compared to NB muscles. The WB syndrome had a great effect on the texture and connective tissue properties of the meat compared to normal muscle, with a tendency for having a lower water holding capacity and higher raw meat hardness

ECM provides various mechanical cues that are able to affect the self‑renewal and differentiation of mesenchymal stem cells (MSC). Little is known however how these ques work in a pathological environment, such as acute oxidative stress. To better understand the behavior of human adipose tissue-derived MSC (ADMSC) in such conditions here we provide morphological and quantitative evidence for significantly altered early steps of mechanotransduction when adhering to oxidized collagen (Col-Oxi). This affects both focal adhesion (FA) formation and YAP/TAZ signaling events. Representative morphological images show that ADMSCs spread better within 2 h of adhesion on native collagen (Col), while they tended to round up on Col-Oxi. It correlates also with the less development of the actin cytoskeleton and FA formation, confirmed quantitatively by morpho-metric analysis using ImageJ. As shown by immunofluorescence analysis, oxidation also affected the ratio of cytosolic to nuclear YAP/TAZ activity, concentrating in the nucleus for Col while remaining in the cytosol for Col-Oxi, suggesting abrogated signal transduction. Comparative AFM studies show that native collagen forms relatively coarse aggregates, much thinner with Col-Oxi, possibly reflecting its altered ability to aggregate. On the other hand, the corresponding Young's moduli were only slightly changed, so viscoelastic properties cannot explain the observed biological differences. However, the roughness of the protein layer decreases dramatically, from RRMS equal to 27.95 + 5.1 nm for Col to 5.51 + 0.8 nm for Col-Oxi (p<0.05), which dictates our conclusion that it is the most altered parameter in oxidation. Thus, it appears to be a predominantly topo-graphic response that affects the mechanotransduction of ADMSCs by oxidized collagen.

Cartilage development is a tightly regulated process that involves multiple molecules and signaling pathways. The loss of expression of a single gene can alter cell behavior and disrupt the development of the cartilage. Col11a1 encodes the alpha one chain of the minor fibrillar collagen type XI that is essential in skeletal development. We used an RNAi mediated knockdown approach to investigate the role of Col11a1 expression during chondrogenesis in ATDC5 cells. Col11a1 expression promotes the transition of mesenchymal cells to the chondrogenic phenotype, and reduction of Col11a1 expression interferes with cellular differentiation. Our results indicate that collagen α1(XI) protein is required for chondrocyte cell shape, matrix production, mineralization and gene expression through a mechanism that involves AKT/GSK3β/β-catenin and TCF/LEF activity in ATDC5 cells.

Currently, the clinical impact of cell therapy after a myocardial infarction (MI) is limited by low cell engraftment due to significant cell death, including apoptosis, in an infarcted, inflammatory, poor angiogenic environment, low cell retention and secondary migration. Cells interact with their environment through integrin mechanoreceptors that control their survival/apoptosis/differentiation/migration/proliferation. Optimizing these interactions may be a way of improving outcomes. The association of free cells with a 3D-scaffold may be a way to target their integrins. Collagen is the most abundant structural component of the extracellular matrix (ECM) and the best contractility levels are achieved with cellular preparations containing collagen, fibrin, or Matrigel (i.e. tumor extract). In the interactions between cells and ECM, 3 main proteins are recognised: collagen, laminin and RGD (Arg-Gly-Asp) peptide. The RGD plays a key role in heart development, after MI, and on cardiac cells. Cardiomyocytes secrete their own laminin on collagen. The collagen has a non-functional cryptic RGD and is thus suboptimal for interactions with associated cells. The use of a collagen functionalized with RGD may help to improve collagen biofunctionality. It may help in the delivery of paracrine cells, whether or not they are contractile, and in assisting tissue engineering a safe contractile tissue.

Recently, two chicken breast fillet abnormalities, termed Wooden Breast (WB) and Spaghetti Meat (SM), have become a challenge for the chicken meat industry. The two abnormalities share some overlapping morphological features, including myofiber necrosis, intramuscular fat deposition, and collagen fibrosis, but display very different textural properties. WB has a hard, rigid surface, while the SM has a soft and stringy surface. Connective tissue is affected in both WB and SM, and accordingly, this study's objective was to investigate the major component of connective tissue, collagen. The collagen structure was compared with normal (NO) fillets using histological methods and Fourier transform infrared (FTIR) microspectroscopy and imaging. The histology analysis demonstrated an increase in the amount of connective tissue in the chicken abnormalities, particularly in the perimysium. The WB displayed a mixture of thin and thick collagen fibers, whereas the collagen fibers in SM were thinner, fewer, and shorter. For both, the collagen fibers were oriented in multiple directions. The FTIR data showed that WB contained more β-sheets than the NO and the SM fillets, whereas SM fillets expressed the lowest mature collagen fibers. This insight into the molecular changes can help to explain the underlying causes of the abnormalities.

Tenascin C (TNC) is an element of the extracellular matrix (ECM) of various tissues, including the skin, and is involved in modulating ECM integrity and cell physiology. Although skin aging is apparently associated with changes in the ECM, little is known about the role of TNC in skin aging. Here we found that Tnc mRNA level was significantly reduced in the skin tissues of aged mice compared with young mice, consistent with reduced TNC protein expression in aged human skin. TNC-large (TNC-L; 330-kDa) and -small (TNC-S; 240-kDa) polypeptides were observed in conditional media from primary dermal fibroblasts. Both recombinant TNC polypeptides, corresponding to TNC-L and TNC-S, increased the expression of type I collagen and reduced the expression of matrix metalloproteinase-1 in fibroblasts. Treatment of fibroblasts with a recombinant TNC polypeptide, corresponding to TNC-L, induced phosphorylation of SMAD2 and SMAD3. TNC increased the level of TGF-β1 mRNA and upregulated the expression of type I collagen by activating the TGF-β signaling pathway. In addition, TNC also promoted the expression of type I collagen in fibroblasts embedded in a three-dimensional collagen matrix. Our findings suggest that TNC contributes to the integrity of ECM in young skin and to prevention of skin aging.

Accumulation of damage is generally considered the cause of aging. Interventions that delay aging mobilize mechanisms that protect and repair cellular components. Consequently, research has been focused on studying the protective and homeostatic mechanisms within cells. However, in humans and other multicellular organisms, cells are surrounded by extracellular matrices (ECM), which are important for tissue structure, function and intercellular communication. During aging, components of the ECM become damaged through fragmentation, glycation, crosslinking, and accumulation of protein aggregation, all of which contribute to age-related pathologies. Interestingly, placing senescent cells into a young ECM rejuvenates them and we found that many longevity-assurances pathways re-activate de-novosynthesis of ECM proteins during aging. This raises the question of what constitutes a young ECM to reverse aging or maintain health? In order to make inroads to answering this question, I suggest a systems-level approach of quantifying the matrisome or ECM compositions reflecting health, pathology, or phenotype and propose a novel term, the “matreotype”, to describe this. The matreotype is defined as the composition and modification of ECM or matrisome proteins associated with or caused by a phenotype, such as longevity, or a distinct and acute physiological state, as observed during aging or disease. Every cell type produces its unique ECM. Interestingly, cancer-cell types can even be identified based on their unique ECM composition. Thus, the matreotype reflects cellular identity and physiological status. Defined matreotypes could be used as biomarkers or prognostic factors for disease or health status during aging with potential relevance for personalized medicine. Treatment with biologics that alter ECM-to-cell mechanotransduction might be a strategy to reverse age-associated pathologies. An understanding of how to reverse from an old to a young matreotype might point towards novel strategies to rejuvenate cells and help maintain tissue homeostasis to promote health during aging.

Three-dimensional matrices are a new strategy used to tackle type I diabetes; a chronic metabolic disease characterized by the destruction of beta pancreatic cells. Type I collagen is an abundant extracellular matrix (ECM), component that has been used to support cell growth. However, pure collagen possesses some difficulties including low stiffness and strength, and high susceptibility to cell-mediated contraction. Therefore, we developed a collagen hydrogel with a poly(ethylene glycol) diacrylate (PEGDA) interpenetrating network (IPN), functionalized with vascular endothelial growth factor (VEGF) to mimic the pancreatic environment for the sustenance of beta-pancreatic cells. We analyzed the physicochemical characteristic of the hydrogels and found that they were successfully synthesized. The mechanical behavior of the hydrogels improved with the addition of VEGF, and the swelling degree and the degradation were stable over time. In addition, it was found that 5 ng/mL VEGF-functionalized collagen/PEGDA IPN hydrogels sustained and enhanced viability, proliferation, respiratory capacity and functionality of beta pancreatic cells. Hence, this is a potential candidate for future preclinical evaluation, which may be favorable for diabetes treatment.

During the progression from ductal carcinoma in situ (DCIS) to invasive breast cancer (IBC), cells have to overcome the physically restraining basement membrane (BM) which compartmentalizes the epithelium from the stroma. Since the extracellular matrix (ECM) of the epithelial and stromal compartment is biochemically and physically distinct from one another, the progression demands a certain degree of cellular plasticity being essentially required for a primary tumor to become invasive. The Epithelial-to-Mesenchymal Transition depicts such a cell program equipping cancer cells with features allowing for dissemination from the epithelial entity and stromal invasion on the single-cell level. Here, we investigated the reciprocal interference between an altering tumor microenvironment and the EMT-phenotype in vitro. BM-typical collagen IV and stroma-typical collagen I coatings were applied as provisional 2-D matrices. Pro-inflammatory growth factors were introduced to improve tissue mimicry. Whereas the growth on coated surfaces did only slightly affect the EMT-phenotype, the combinatorial action of collagen with growth factor TGF-β1 induced prominent phenotypic changes. However, the EMT-induction was independent of the collagen type and cellular accessibility for EMT-like changes was strongly cell line dependent. Summarizing the entire body of data, we computed an EMT-phenotyping model that was used to decide on cellular EMT-status and estimate EMT-like changes. We confirmed that miR200c-mediated reversion of mesenchymal MDA-MB-231 cells is reflected by our EMT-phenotype model emphasizing its potential to predict the therapeutic efficacy of EMT-targeting drugs in the future.

To resolve the controversial issues of UV-light-initiatedcornealcollagen cross-linking (CXL) by theoretical formulas and measured clinical outcomes. The controversial issues are addressed and resolved by analytical formulas including: the validation of Bunsen Roscoe law (BRL), the cutoff light intensity, the minimum corneal thickness, the demarcation line depth, the role of oxygen riboflavin (RF) concentration. The overall CXL efficacy is governed by UV-A light intensity, dose, exposure time, mode of exposure (pulsed or CW), riboflavin concentration, diffusion and drops pre-operation and interoperation administration, concentration of oxygen in the stromal tissue (pre-op and inter-op), and environmental conditions. The length of the riboflavin presoaking time and viscosity of the riboflavin film also affect the crosslink depth. Analytic formulas are derived for the scaling laws for type-I and type-II efficacy, given by the square-root of light intensity, and light dose, respectively. The controversial issues of CXL may be partially resolved via analytic formulas, and compared with measurements. The scaling laws of type-I and type-II efficacy are different and given by analytic formulas. Our formulas also predict the maximum light intensity and the minimum corneal thickness, which are consistent with measurements.

Collagen is the most abundant protein in the animal kingdom. Industrial collagen is mainly bovine and porcine origin. However, due to religious beliefs, allergic issues, and infectious diseases, alternative sources of collagen as marine are gaining increasing interest. In this work, the acid-soluble collagen (ASC) were extracted from salt-cured Atlantic cod (Gadus morhua) skin and characterized. The extraction yield was about 2.0%, equivalent to the extraction yield reported for other fish skins. The electrophoretic pattern showed the typical type I structure (α, β and γ chains). UV-VIS and FTIR absorbance spectra suggested a very pure ASC with an intact triple helical structure. The integrity and the adequate porosity required for different applications were then confirmed by electron micrograph. Our findings allow us to say that, for the first time, we extracted acid-soluble type I collagen from salt-cured Atlantic cod skin, with characteristics suitable for application in various fields, such as biomedical.

Though melatonin is known to improve ultraviolet B (UVB)-induced oxidative damage and inflammatory conditions via blockade of nuclear factor (NF)-κB, interleukin (IL)-6, there is no report on anti-wrinkle effect of melatonin to date. Hence in the present study, anti-wrinkle mechanism of melatonin was elucidated in UVB treated HaCaT keratinocytes and hairless mice. Herein melatonin protected against a radical initiator tert-Butyl hydroperoxide (t-BOOH) induced reactive oxygen species (ROS) production, matrix metalloprotease 1 (MMP-1) and cytotoxicity in HaCaT keratinocytes. Also, melatonin suppressed the expression of sonic hedgehog (SHH) and GLI for hedgehog signaling, p-NF-kB, cyclooxygenase (COX-2), p-ERK for inflammatory responses in UVB treated HaCaT keratinocytes. Furthermore, melatonin protected skin from wrinkle formation, transdermal water loss in hairless mice irradiated by UVB for 8 weeks. Notably, melatonin prevented against epidermal thickness and dermal collagen degradation in UVB irradiated hairless mice by Hematoxylin & Eosin and Masson’s trichrome staining. Taken together, these findings suggest that melatonin reduces wrinkle formation via inhibition of ROS/SHH and inflammatory proteins such as NF-kB/COX-2/ERK/MMP1.

The treatment of large-area bone defects remains a challenge; however, various strategies have been developed to improve the performances of scaffolds in bone tissue engineering. In this study, poly(lactide-co-glycolide)/hydroxyapatite (PLGA/HA) scaffold was coated with Asp-Gly-Glu-Ala (DGEA)-incorporated collagen for the repair of rat skull defect. Our results indicated that the mechanical strength and hydrophilicity of PLGA/HA scaffold were clearly improved and conducive to cell adhesion and proliferation. The collagen-coated scaffold with DGEA significantly promoted the repair of skull defect. These findings indicated that a combination of collagen coating and DGEA improved scaffold properties for bone regeneration, thereby providing a new potential strategy for scaffold design.

Since the past few decades, numerous modifications and innovations have been done to design the optimal corneal biomatrix for corneal epithelial cells (CECs) or limbal epithelial stem cells (LESCs) carriers. However, researchers have yet to discover the ideal optimization strategies in the development of corneal biomatrix design and its effects on cultured CECs or LESCs. This review further discusses and summarizes recent optimization strategies to develop an ideal collagen biomatrix and its interaction with CECs and LESCs. Using PRISMA guidelines, the articles published from June 2012 to June 2022 were systematically searched using Web of Science (WoS), Scopus, PubMed, Wiley, and EBSCOhost databases. The literature search identified 444 potential relevant published articles, with 29 relevant articles selected based on the inclusion and exclusion criteria after the screening and appraising processes. The current paper highlights the physicochemical and biocompatibility (in vitro and in vivo) characterization methods, which were inconsistent throughout the different studies. Despite the variability in the methodology approach, the reviewer postulated that the modification of the collagen biomatrix improves its mechanical and biocompatibility properties toward CECs and LESCs. All findings were discussed in this review; thus, it provides a general view of up-to-date trends in this field.

Mesenchymal stem cells (MSCs) are involved in the process of extracellular matrix (ECM) remodeling where collagens play a pivotal role. We recently demonstrated that the remodeling of adsorbed collagen type I might be disordered upon oxidation following its fate in the presence of human adipose-derived MSC (ADMSCs). With the present study, we intended to learn more about the effect of polyphenolic antioxidant Epigallocatechin gallate (EGCG) attempting to mimic the conditions of oxidative stress in vivo and its putative prevention by antioxidants. Collagen Type I was isolated from mouse tail tendon (MTC) and labeled with FITC before oxidizing according to Fe2+/H2O2 protocol. FITC-collagen remodeling by ADMSC was assessed morphologically before and after EGCG pretreatment and confirmed via detailed morphometry analysis measuring the anisotropy index (AI) and fluorescence intensity (FI) in selected regions of interest (ROI), namely: outside the cells; over the cells and central (nuclear perinuclear) region, whereas the pericellular proteolytic activity was measured by de-quenching of fluorescent collagen probes (FRET effect). Here we provide morphological evidence that MTC undergoes significant reorganization by the adhering ADMSC along with the substantial activation of pericellular proteolysis, and further confirm that both processes are suppressed upon collagen oxidation. An important observation was that this abrogated remodeling cannot be prevented by the EGCG pretreatment. Conversely, the detailed morphometry analysis showed that oxidized FITC-collagen rather tends to accumulate beneath the cells and around the cell’s nuclei suggesting the activation of alternative routes for its removal, such as internalization and/or transcytosis. Morphometry analysis also revealed that both processes are supported by EGCG pretreatment.

Freeze-drying is a well-established process in biomedical engineering for the fabrication of three-dimensional open-porous medical devices, especially those based on biopolymers. One of the most used biopolymers in this field is collagen, the most abundant protein in the human body and the main component of the extracellular-matrix, as well as its derivatives. Freeze-dried collagen-based sponges with a wide variety of attributes can be produced by design and have led to a wide range of successful commercial medical devices, foremost for dental, orthopedic, hemostatic and neuronal applications. However, this is still considered a high-cost and time-consuming process that is often used in a non-optimized manner. By combining advances in other technological fields, the opportunity arises to further evolve this process in a sustainable manner, and optimize the resulting products as well as create new opportunities in this field.

Meat tenderness, water holding capacity (WHC) and colour attributes of six muscles (Longissimus thoracis et lumborum (LTL), Semimembranosus (SM), Biceps femoris (BF), Supraspinatus (SS), Infraspinatus (IS), Semitendinosus (ST)) from large frame Indigenous Veld Goats (IVG) and Boer Goats (BG) were studied. Weaner male Boer Goats (BG; n = 18; 10 bucks and 8 wethers) and large frame Indigenous Veld Goats (IVG; n = 19; 9 bucks and 10 wethers) were raised on hay and natural grass, and on a commercial pelleted diet to a live weight of 30 - 35 kg. All goats were slaughtered at a commercial abattoir and the dressed carcasses chilled at 4°C within 1-hour post-mortem. The muscles were dissected from both sides 24-hours post-mortem and aged for 1-day and 4-days. Variations in meat characteristics such as ultimate pH, WHC, percentage purge, myofibril fragment length, intramuscular fat, connective tissue characteristics, and Warner-Bratzler shear force. Bucks had higher L* and Hue-angle values, whereas wethers had increased a* and Chroma values. The muscle baseline-data will allow informed decisions to support muscle-specific marketing strategies, which may be used to improve consumer acceptability of chevon.

Malnutrition is one of major factors of bone and cartilage disorders. Pacific cod (Gadus macrocephalus) processing waste is a cheap and highly promising source of bioactive substances, including collagen-derived peptides and amino acids, for bone and cartilage structure stabilization. Addition of these substances to a functional drink is one of the ways to achieve their fast intestinal absorption. Collagen hydrolysate was obtained via enzymatic hydrolysis, ultrafiltration, freeze-drying, and grinding to powder. The lyophilized hydrolysate was a light gray powder with high protein content (>90%), including collagen (about 85% of total protein) and a complete set of essential and non-essential amino acids. The hydrolysate was applicable as a protein food supply or a structure-forming food component due to presence of collagen fiber fragments. An isotonic fitness drink (osmolality 298.1 ± 2.1 mOsm/L) containing the hydrolysate and vitamin C as a cofactor in collagen biosynthesis was prepared. Addition of the hydrolysate did not adversely affect its organoleptic parameters. Production of such functional foods and drinks is one of the beneficial ways of fish processing waste utilization.

Therapeutic resistance in pancreatic ductal adenocarcinoma (PDAC) can be attributed, in part, to a dense extracellular matrix containing excessive collagen deposition. Here, we describe a novel Salmonella typhimurium (ST) vector expressing the bacterial collagenase Streptomyces omiyaensis trypsin (SOT), a serine protease known to hydrolyze collagens I and IV, which are predominantly found in PDAC. Utilizing aggressive models of PDAC, we show that ST-SOT selectively degrades intratumoral collagen leading to enhancement of immune checkpoint blockade (ICB) therapy in tumor-bearing mice. Ultimately, we found that ST-SOT treatment significantly modifies the intratumoral immune landscape to generate a microenvironment more conducive to ICB.

Collagen has been accepted in drug delivery structures due to its biocompatible chemical, physicochemical and curative actions. The study aims to evaluate the effects of a bovine collagen hydrolysate (Clg) on the main photophysical and photodynamic properties of a new gallium (III) phthalocyanine (GaPc). The absorbance of GaPc and the conjugate GaPc-Clg showed a decrease of the intensive Q-band (681 nm) and a blue shift of the maximum (678 nm) with loss of UV-band (354 nm) for 40 mg/mL Clg. The fluorescence of GaPc was evaluated with strong emission peak at 694 nm (exc: 615 nm) which was lower for GaPc-Clg (691 nm). The quantum yield of 0.23 was obtained for GaPc which was reduced for GaPc-Clg (0.012). Photo- and dark cytotoxicity was studied by NRU-assay on pigmented melanoma cells (SH-4) and normal cell lines (BJ and HaCaT). The results suggested a slight reduction of cytotoxicity for the conjugate but almost double for the selectivity index (SI: 0.71 vs. 1.49 for HaCaT cells). The study showed that bovine collagen hydrolysate can lower the PDT activity of a powerful photosensitizer with phototherapeutic index: 819 to 360, but also minimized its dark toxicity as a positive effect in the photodynamic treatment.

The bioactivities of collagen-hydrolysates, sulfated glucosamine and a special fatty acid enriched dog-food were tested in a dog patient study as potential therapeutic treatment options in early osteoarthritis. Biophysical, biochemical, cell biological and molecular modeling methods support that these well-defined substances may act as effective nutraceuticals. Importantly, the applied collagen-hydrolysates as well as sulfated glucosamine residues from marine organisms were strongly supported by both an animal model and molecular modeling of intermolecular interactions. Molecular modeling of predicted interaction dynamics were evaluated for the receptor proteins MMP-3 and ADAMTS-5. These proteins play a prominent role in the maintenance of cartilage health as well as innate and adapted immunity. Nutraceuticals data were generated in a veterinary clinical study focusing on mobility and agility. Specifically, key clinical parameters were obtained from blood probes of German shepherd dogs with early osteoarthritis symptoms fed with collagen-hydrolysates or sulfated glucosamines. Collagen-hydrolysate, a chondroprotective food supplement was examined by high resolution NMR experiments. Molecular modeling simulations were used to further characterize the interaction potency of collagen-fragments and glucosamines with protein receptor structures. Potential beneficial effects of collagen-hydrolysates, sulfated glycans (i.e. sulfated glucosamine from crabs and mussels) and lipids, especially, eicosapentaenoic acid (extracted from fish oil) on biochemical and physiological processes are discussed here in the context of human and veterinary medicine.

Downregulated microRNA-142-3p signaling contributes to the pathogenesis of endometriosis [1] [2], an invasive disease where the lining of the uterus grows at ectopic locations, by yet incompletely understood mechanisms. Using bioinformatics and in vitro assays, this study identifies cytoskeletal regulation and integrin signaling as two relevant categories of miR-142-3p targets. qPCR revealed that miR-142-3p upregulation in St-T1b cells downregulates ROCK2, CFL2, RAC1, WASL and ITGAV. qPCR and Western-blotting showed miR-142-3p effect on WASL and ITGAV was significant also in primary endometriotic stroma cells. Luciferase reporter assays in ST-T1b cells then confirmed direct regulation of ITGAV and WASL. On the functional side, miR-142-3p upregulation significantly reduced ST-T1b cell size, the size of vinculin plaques, migration through fibronectin-coated transwell filters and the ability of ST-T1b and primary endometriotic stroma cells to contract collagen I gels. These results suggest that miR-142-3p has a strong mechanoregulatory effect on endometrial stroma cells and its external administration reduces the invasive endometrial phenotype. Within the limits of an in vitro investigation, our study provides new mechanistic insights into the pathogenesis of endometriosis and provides a perspective for the development of miR-142-3p based drugs for inhibiting invasive growth of endometriotic cells.

Wounds represents a medical problem that contribute importantly to patient morbidity and to the healthcare costs in several pathologies. In Hidalgo, Mexico, Bacopa procumbens plant has been traditionally used for wound healing care for several generations; in vitro and in vivo experiments were design to evaluate the effects of bioactive compounds obtained from B. procumbens aquoethanolic extract and to determine the key pathways involved in wound regeneration. Bioactive compounds were characterized by HPLC- QTOF-MS and proliferation, migration, adhesion, and differentiation studies were done on NIH/3T3 fibroblasts. Polyphenolic compounds from Bacopa procumbens (PB) regulated proliferation and cell adhesion; enhanced migration reducing the artificial scratch area; and modulated cell differentiation. PB compounds were included in a hydrogel for topical administration on rat excision wound model. Histological, histochemical and mechanical analysis showed that PB treatment accelerates wound closure in at least 48 h; reduce inflammation, increasing cell proliferation and deposition and organization of collagen in earlier times. These changes resulted in the formation of a scar with better tensile properties. Immunohistochemistry and RT-PCR molecular analyses demonstrated that treatment induces: i) overexpression of transforming growth factor beta (TGF-β); and ii) the phosphorylation of Smad 2/3 and ERK1/2, suggesting the central role of some PB to enhance wound healing, modulating TGF-β activation.

Rheumatoid arthritis (RA) is a chronic autoimmune disease, causing inflammation of joints, cartilage destruction and bone erosion. Biomarkers and new drug targets are actively sought and progressed to improve available options for patient treatment. The Collagen Triple Helix Repeat Containing 1 protein (CTHRC1) may have an important role as a biomarker for rheumatoid arthritis, as CTHRC1 protein concentration is significantly elevated in the peripheral blood of rheumatoid arthritis patients, compared to osteoarthritis (OA) patients and healthy individuals. CTHRC1 is a secreted glycoprotein that promotes cell migration and has been implicated in arterial tissue-repair processes. Furthermore, high CTHRC1 expression is observed in many types of cancer and this is associated with cancer metastasis to the bone and poor prognosis. However, the function of CTHRC1 in RA is still largely undefined. The aim of this review is to summarize recent findings on the role of CTHRC1 as a potential biomarker and pathogenic driver of RA progression that may be linked to the pathogenic behavior of fibroblast-like synoviocytes, cartilage destruction, and bone erosion.

Here we report the preparation and characterization of a novel biomimetic toothpaste containing morphogenetically active amorphous polyphosphate (polyP) microparticles enriched with retinyl acetate (“a-polyP/RA-MP”). The spherical microparticles (average size, 550±120 nm), prepared by co-precipitating sodium-polyP with calcium chloride and supplemented with retinyl acetate, were incorporated into a basis toothpaste at a final concentration of 1% or 10%. The paste containing “a-polyP/RA-MP” significantly increased the growth of human mesenchymal stem cells (MSC), compared to a commercial toothpaste which acts rather inhibitory and the paste without polyP and retinyl acetate. qRT-PCR experiments revealed that the retinoid causes an induction of the expression of the MSC marker genes for osteoblast differentiation encoding collagen type I and alkaline phosphatase. On the other hand, the polyP ingredient, supplied as Zn-polyP microparticles (“Zn-a-polyP-MP”) strongly inhibited the growth of the cariogenic bacterium Streptococcus mutans. We demonstrate that the amorphous polyP-containing toothpaste, enriched with retinyl acetate, efficiently repairs both cracks/fissures and carious lesions in the tooth enamel, and reseals dentinal tubules, already after a 5 d treatment (brushing) of teeth twice daily for 5 min as examined by SEM and quantitative EDX analysis. The stability of the occlusion of dentin cracks even turned out to resist against short high power sonication treatment. Our results demonstrate that the novel toothpaste prepared here, containing amorphous polyP and retinyl acetate, is particularly suitable for prevention/repair of (cariogenic) damages of tooth enamel/dentin and for treatment of dental hypersensitivity.

The extracellular matrix (ECM) is earning an increasingly relevant role in many disease states and the process of aging. Analyzing these disease states is possible with GWAS and PheWAS methodology, and through our analysis, we aimed to explore the relationships between polymorphisms in the compendium of ECM genes (i.e., matrisome genes) in various disease states. A significant contribution on the part of the ECM polymorphisms is evident in many varying types of diseases, particularly those in the core matrisome genes. Our results confirm previous links to connective tissue disorders, but also unearth new and underexplored relationships with neurological, psychiatric, and age-related disease states. Upon analysis of drug indications for gene-disease relationships, we identified numerous targets that may be repurposed for age-related pathology. The identification of ECM polymorphisms and their contribution to disease plays an integral role in future therapeutic developments, drug repurposing, precision medicine, and personalized care.

Recombinant human collagen peptide (RCP) is a recombinantly created xeno-free biomaterial enriched in RGD (arginine-glycine-aspartic acid) sequences, with good processability that is being investigated for regenerative medicine applications. Recently, the biocompatibility and osteogenic ability of β-TCP/RCP (RCP granules combined with β-tricalcium phosphate (TCP) submicron particles) were demonstrated. In the present study, β-TCP/RCP was implanted into experimental periodontal tissue defects (three-walled bone defect) created in beagle dogs to investigate tissue responses and subsequent regenerative effects. Micro computed tomography image analysis at 8 weeks postoperatively showed that the amount of new bone after β-TCP/RCP graft was significantly greater (2.2 fold, P&lt;0.05) than that of the control (no graft) group. Histological findings showed that the transplanted β-TCP/RCP induced active bone-like tissue formation including TRAP-positive and OCN-positive cells as well as bioabsorbability. Ankylosis did not occur, and periostin-positive periodontal ligament-like tissue formation was observed. Histological measurements revealed that β-TCP/RCP implantation formed 1.7-fold more bone-like tissue and 2.1-fold more periodontal ligament-like tissue than the control, and significantly suppressed gingival recession and epithelial downgrowth (P&lt;0.05). These results suggest that β-TCP/RCP is effective as a periodontal tissue regenerative material.