Collagen Sponge Research Articles

Hemostatic Collagen Sponge with High Porosity Promotes the Proliferation and Adhesion of Fibroblasts and Osteoblasts.

The use of biomaterial for tissue repair involves the interaction between materials and cells, and the coagulum formation represents the first step of tissue healing. This process is particularly critical in the oral cavity, where the wounds are immediately subjected to the masticatory mechanical stress, saliva invasion, and bacterial attack. Therefore, the present study aimed to explore the structural features and the biological activities of a hemostatic collagen sponge on human gingival fibroblasts (HGFs) and human oral osteoblasts (HOBs). The microstructure of the collagen sponge was characterized by a scanning electron microscope (SEM) and histological analysis. The porosity was also calculated. To investigate biological activities, HGFs and HOBs were cultured on the collagen sponges, and their adhesion was observed at SEM on the third day, while cell viability was investigated at the third and seventh days by Tetrazolium (MTT) assay. For osteoblasts seeded on collagen sponge the mineralization ability was also evaluated by alkaline phosphatase (ALP) assay at the seventh day, and by Alizarin red staining on the 14th. Furthermore, the gene expression of ALP and osteocalcin (OCN) was investigated after 3, 7 and 14 days. SEM images of the sponge without cells showed a highly porous 3D structure, confirmed by the measurement of porosity that was more than 90%. The samples cultured were characterized by cells uniformly distributed and adhered to the sponge surface. Proliferation ended up being promoted, as well as the mineralization ability of the osteoblasts, mainly at the mature stage. In conclusion, this collagen sponge could have a potential use for tissue healing.

Treatment with gentamicin-impregnated collagen sponges in reducing infection of implantable cardiac devices: 10-year analysis with propensity score matching

Introduction and ObjectivesThe incidence of device infection has increased over time and is associated with increased mortality in patients with cardiac implantable electronic devices (CIEDs). Gentamicin-impregnated collagen sponges (GICSs) are useful in preventing surgical site infection (SSI) in cardiac surgery. Nevertheless, to date, there is no evidence concerning their use in CIED procedures. Our study aims to determine the effectiveness of treatment with GICSs in preventing CIED infection. MethodsA total of 2986 adult patients who received CIEDs between 2010 and 2020 were included. Before device implantation, all patients received routine periprocedural systemic antibiotic prophylaxis. The study endpoints were the CIED infection rate at one year and the effectiveness of the use of GICSs in reducing CIED infection. ResultsAmong 1524 pacemaker, 942 ICD and 520 CRT implantations, CIED infection occurred in 36 patients (1.2%). Early reintervention (OR 9 [95% CI 3.180–25.837], p<0.001), pocket hematoma (OR 11 [95% CI 4.195–28.961], p<0.001), diabetes (OR 2.9 [95% CI 1.465–5.799], p=0.002) and prolonged procedural time (OR 1.02 [95% CI 1.008–1.034], p=0.001) were independent risk factors for CIED infection. Treatment with GICSs reduced CIED infections significantly ([95% CI −0.031 to −0.001], p<0.001). ConclusionsThe use of GICSs may help in reducing infections associated with CIED implantation.

EXPLOITING NOVEL 3D JELLYFISH COLLAGEN SPONGES FOR BONE TISSUE REGENERATION

Jellyfish collagens exhibit auspicious perspectives for tissue engineering applications primarily due to their outstanding compatibility with a wide range of cell types, low immunogenicity and biodegradability. Furthermore, derived from a non-mammalian source, jellyfish collagens reduce the risk of disease transmission, minimising therefore the ethical and safety concerns. The current study aims to investigate the potential of 3-dimensional jellyfish collagen sponges (3D-JCS) in promoting bone tissue regeneration.Both qualitative and quantitative analyses were performed in order to assess adhesion and proliferation of MC3T3 cells on 3D-JCL, as well as cell migration and bone-like ECM production. Histological and fluorescent dyes were used to stain mineral deposits (i.e. Alizarin Red S (ARS), Von Kossa, Tetracycline hydrochloride) while images were acquired using optical and confocal microscopy.Qualitative data indicated successful adhesion and proliferation of MC3T3 cells on the 3D-JCS as well as cell migration along with ECM production both on the inner and outer surface of the scaffolds. Moreover, quantitative analyses indicated a four-fold increase of ARS uptake between 2- and 3-dimensional cultures (N=3) as well as an eighteen-fold increase of ARS uptake for the 3D-JCS (N=3) when cultured in osteogenic conditions compared to control. This suggests the augmented osteogenic potential of MC3T3 cells when cultured on 3D-JCS. Nevertheless, the cell-mediated mineral deposition appeared to alter the mechanical properties of the jellyfish collagen sponges that were previously reported to exhibit low mechanical properties (compressive modulus: 1-2 kPa before culture).The biocompatibility, high porosity and pore interconnectivity of jellyfish collagen sponges promoted adhesion and proliferation of MC3T3 cells as well as cell migration and bone-like ECM production. Their unique features recommend the jellyfish collagen sponges as superior biomaterial scaffolds for bone tissue regeneration. Further studies are required to quantify the change in mechanical properties of the cell-seeded scaffolds and confirm their suitability for bone tissue regeneration. We predict that the 3D-JCS will be useful for future studies in both bone and bone-tendon interface regeneration.AcknowledgmentsThis research has been supported by a Medical Research Scotland Studentship award (ref: -50177-2019) in collaboration with Jellagen Ltd.

BIOMIMETIC HEMATOMA: NOVEL CARRIER DELIVERS EXTREMELY LOW-DOSE RHBMP-2 FOR HIGHLY EFFECTIVE HEALING OF LARGE BONE DEFECTS IN GOATS

Our previous rat study demonstrated an ex vivo-created “Biomimetic Hematoma” (BH) that mimics the intrinsic structural properties of normal fracture hematoma, consistently and efficiently enhanced the healing of large bone defects at extremely low doses of rhBMP-2 (0.33 μg). The aim of this study was to determine if an extremely low dose of rhBMP-2 delivered within BH can efficiently heal large bone defects in goats.Goat 2.5 cm tibial defects were stabilized with circular fixators, and divided into groups (n=2-3): 2.1 mg rhBMP-2 delivered on an absorbable collagen sponge (ACS); 52.5 μg rhBMP-2 delivered within BH; and an empty group. BH was created using autologous blood with a mixture of calcium and thrombin at specific concentrations. Healing was monitored with X-rays. After 8 weeks, femurs were assessed using microCT.Using 2.1 mg on ACS was sufficient to heal 2.5 cm bone defects. Empty defects resulted in a nonunion after 8 weeks. Radiographic evaluation showed earlier and more robust callus formation with 97.5 % (52.5 μg) less of rhBMP-2 delivered within the BH, and all tibias were fully bridged at 3 weeks. The bone mineral density was significantly higher in defects treated with BH than with ACS. Defects in the BH group had smaller amounts of intramedullary and cortical trabeculation compared to the ACS group, indicating advanced remodeling.The results confirm that the delivery of rhBMP-2 within the BH was much more efficient than on an ACS. Not only did the large bone defects heal consistently with a 40x lower dose of rhBMP-2, but the quality of the defect regeneration was also superior in the BH group. These findings should significantly influence how rhBMP-2 is delivered clinically to maximize the regenerative capacity of bone healing while minimizing the dose required, thereby reducing the risk of adverse effects.

Evaluation of Effectiveness of New Samples of Chitosan-Based Local Hemostatic Agents After Liver Resection in Experiment

INTRODUCTION: Chitosan-based local hemostatic agents are most promising in terms of effective stoppage of bleeding, additional properties (for example, antibacterial effect) and stimulation of regeneration. New forms of them are being developed for different types of organ damage.&#x0D; AIM: To evaluate the hemostatic effect of samples of new chitosan-based local hemostatic agents on a liver resection model.&#x0D; MATERIALS AND METHODS: An in vivo experiment was performed on 60 white male rats of Wistar line of 200 g250 g mass. The animals were divided to 4 study groups of 15 animals, respectively, depending on the kind of hemostatic agent and additional introduction of an anticoagulant that enhanced bleeding. As study materials, hemostatic collagen sponge (control groups No. 1.1 and 1.2) and also samples of new chitosan-based hemostatic agents Сhitocol-Hemo (Evers, Russia) were used. The rats under general anesthesia underwent midline laparotomy followed by laparopexy by dissecting the falciform ligament of the liver and placement of a gauze turunda between the diaphragm and the left liver lobule with displacement of the latter into the wound. After this, a sterile gauze turunda of the known mass was placed under the left lateral lobe of the liver, and resection of this lobe was performed at 10 mm distance from the edge. The bleeding was stopped by application of the tested materials. The mass of blood loss (gravimetric parameters) and the time of bleeding were evaluated. The reliability of the differences was determined using nonparametric Mann-Whitney test.&#x0D; RESULTS: In animals that were not administered the anticoagulant before modeling of the liver trauma, statistically significant differences were found only in such parameters as increase in the sample mass after impregnation with blood, in percent. Here, the value of this parameter in the group with use of hemostatic collagen sponge (2262.9) was three times that in the group using hemostatic Сhitocol-Hemo (722.7) p = 0.000003. The differences between the groups with heparin therapy were of similar character (p = 000003).&#x0D; CONCLUSION: The hemostatic effect of the sample of Сhitocol-Hemo hemostatic agent was confirmed in an acute experiment on a model of liver injury in rats on the basis of measurement of the mass of blood lost, of blood absorbed by the sample, and also of bleeding time. This hemostatic effect is probably provided due to positive physicochemical characteristics (porous structure, stroma/pores ratio and composition of the agent.

Differentiation of Human Induced Pluripotent Stem Cells into Mature and Myelinating Schwann Cells.

In the peripheral nervous system, Schwann cells (SCs) play a crucial role in axonal growth, metabolic support of neurons, and the production of myelin sheaths. Expansion of SCs after extraction from human or animal nerves is a long and often low-yielding process. We established a rapid cell culture method using a defined serum-free medium to differentiate human induced pluripotent stem cells (iPSCs) into SCs in only 21 days. The SC identity was characterized by expression of SRY-Box Transcription factor 10 (SOX10), S100b, glial fibrillary acidic protein (GFAP), P75, growth-associated protein 43 (GAP43), and early growth response 2 (EGR2) markers. The SC purity reached 87% as assessed by flow cytometry using the specific SOX10 marker, and 69% based on S100b expression. When SCs were cocultured with iPSC-derived motor neurons two-dimensionally or three-dimensionally (3D), they also expressed the markers of myelin MBP, MPZ, and gliomedin. Likewise, when they were seeded on the opposite side of a porous collagen sponge from motor neurons in the 3D model, they were able to migrate through it and colocalize with motor axons after 8 weeks of maturation. Moreover, they were shown by transmission electron microscopy to form myelin sheaths around motor axons. These results suggest that the use of autologous iPSC-derived SCs for clinical applications such as the repair of peripheral nerve damage, the treatment of spinal cord injuries, or for demyelinating diseases could be a valuable option. Impact Statement Peripheral nerve injuries can cause the complete paralysis of the upper or lower limbs, which considerably reduces the quality of life of patients. To repair this injury, many approaches have been developed by tissue engineering. Combining biomaterials with Schwann cells (SCs) has been shown to be an effective solution for stimulating nerve regeneration. However, the challenge faced concerns the strategy for obtaining autologous SCs to treat patients. A promising approach is to differentiate them from the patient's own cells, previously induced into pluripotent stem cells. We propose a fast culture method to generate functional SCs differentiated from induced pluripotent stem cells.

Creation and preclinical evaluation of a novel mussel-inspired, biomimetic, bioactive bone graft scaffold: direct comparison with Infuse bone graft using a rat model of spinal fusion.

Infuse bone graft is a widely used osteoinductive adjuvant; however, the simple collagen sponge scaffold used in the implant has minimal inherent osteoinductive properties and poorly controls the delivery of the adsorbed recombinant human bone morphogenetic protein-2 (rhBMP-2). In this study, the authors sought to create a novel bone graft substitute material that overcomes the limitations of Infuse and compare the ability of this material with that of Infuse to facilitate union following spine surgery in a clinically translatable rat model of spinal fusion. The authors created a polydopamine (PDA)-infused, porous, homogeneously dispersed solid mixture of extracellular matrix and calcium phosphates (BioMim-PDA) and then compared the efficacy of this material directly with Infuse in the setting of different concentrations of rhBMP-2 using a rat model of spinal fusion. Sixty male Sprague Dawley rats were randomly assigned to each of six equal groups: 1) collagen + 0.2 µg rhBMP-2/side, 2) BioMim-PDA + 0.2 µg rhBMP-2/side, 3) collagen + 2.0 µg rhBMP-2/side, 4) BioMim-PDA + 2.0 μg rhBMP-2/side, 5) collagen + 20 µg rhBMP-2/side, and 6) BioMim-PDA + 20 µg rhBMP-2/side. All animals underwent posterolateral intertransverse process fusion at L4-5 using the assigned bone graft. Animals were euthanized 8 weeks postoperatively, and their lumbar spines were analyzed via microcomputed tomography (µCT) and histology. Spinal fusion was defined as continuous bridging bone bilaterally across the fusion site evaluated via µCT. The fusion rate was 100% in all groups except group 1 (70%) and group 4 (90%). Use of BioMim-PDA with 0.2 µg rhBMP-2 led to significantly greater results for bone volume (BV), percentage BV, and trabecular number, as well as significantly smaller trabecular separation, compared with the use of the collagen sponge with 2.0 µg rhBMP-2. The same results were observed when the use of BioMim-PDA with 2.0 µg rhBMP-2 was compared with the use of the collagen sponge with 20 µg rhBMP-2. Implantation of rhBMP-2-adsorbed BioMim-PDA scaffolds resulted in BV and bone quality superior to that afforded by treatment with rhBMP-2 concentrations 10-fold higher implanted on a conventional collagen sponge. Using BioMim-PDA (vs a collagen sponge) for rhBMP-2 delivery could significantly lower the amount of rhBMP-2 required for successful bone grafting clinically, improving device safety and decreasing costs.

Segmental defect healing in the presence or absence of recombinant human BMP2: Novel insights from a rat model.

This study defined and compared the course of native, impaired and growth factor-stimulated bone regeneration in a rat femoral defect model. A mid-diaphyseal defect with rigid internal fixation was surgically created in the right femur of male Fischer rats and serially analyzed over 36 weeks. Native bone regeneration was modeled using a sub-critical, 1 mm size defect, which healed uneventfully. Critical size defects of 5 mm were used to analyze impaired bone regeneration. In a third group, the 5 mm defects were filled with 11 µg of recombinant human bone morphogenetic protein 2 (rhBMP2) impregnated onto an absorbable collagen sponge, modeling its clinical use. Native bone regeneration was characterized by endochondral ossification with progressive remodeling to ultimately resemble intact femora. An endochondral response was also observed under conditions of impaired bone regeneration, but by week 8 medullary capping occurred with fibrofatty consolidation of the tissue within the defect, resembling an atrophic non-union. rhBMP2 treatment was associated with prolonged inflammatory cytokine expression and rapid intramembranous bone formation occurring with reduced expression of cartilage-associated collagens. Between weeks 4 and 36, rhBMP2-treated bones demonstrated decreased trabecular number and increased trabecular separation, which resulted in inferior mechanical properties compared with bones that healed naturally. Clinical Significance: Recombinant human bone morphogenetic protein 2 (rhBMP2) is used clinically to promote healing of long bones. Our data suggest that it drives intramembraneous ossification producing an inferior regenerate that deteriorates with time. Clinical outcomes would be improved by technologies favoring endochondral regenerative ossification.

In vitro glycation of a tissue-engineered wound healing model to mimic diabetic ulcers.

Diabetic foot ulcers are a major complication of diabetes that occurs following minor trauma. Diabetes-induced hyperglycemia is a leading factor inducing ulcer formation and manifests notably through the accumulation of advanced glycation end-products (AGEs) such as N-carboxymethyl-lysin. AGEs have a negative impact on angiogenesis, innervation, and reepithelialization causing minor wounds to evolve into chronic ulcers which increases the risks of lower limb amputation. However, the impact of AGEs on wound healing is difficult to model (both in vitro on cells, and in vivo in animals) because it involves a long-term toxic effect. We have developed a tissue-engineered wound healing model made of human keratinocytes, fibroblasts, and endothelial cells cultured in a collagen sponge biomaterial. To mimic the deleterious effects induced by glycation on skin wound healing, the model was treated with 300 µM of glyoxal for 15 days to promote AGEs formation. Glyoxal treatment induced carboxymethyl-lysin accumulation and delayed wound closure in the skin mimicking diabetic ulcers. Moreover, this effect was reversed by the addition of aminoguanidine, an inhibitor of AGEs formation. This in vitro diabetic wound healing model could be a great tool for the screening of new molecules to improve the treatment of diabetic ulcers by preventing glycation.

Effects and metabolism of fish collagen sponge in repairing acute wounds of rat skin.

Objective: Study the repair effect of tilapia collagen on acute wounds, and the effect on the expression level of related genes and its metabolic direction in the repair process. Materials and methods: After the full-thickness skin defect model was constructed in standard deviation rats, the wound healing effect was observed and evaluated by means of characterization, histology, and immunohistochemistry. RT-PCR, fluorescence tracer, frozen section and other techniques were used to observe the effect of fish collagen on the expression of related genes and its metabolic direction in the process of wound repair. Results: After implantation, there was no immune rejection reaction, fish collagen fused with new collagen fibers in the early stage of wound repair, and was gradually degraded and replaced by new collagen in the later stage. It has excellent performance in inducing vascular growth, promoting collagen deposition and maturation, and re-epithelialization. The results of fluorescent tracer showed that fish collagen was decomposed, and the decomposition products were involved in the wound repair process and remained at the wound site as a part of the new tissue. RT-PCR results showed that, without affecting collagen deposition, the expression level of collagen-related genes was down-regulated due to the implantation of fish collagen. Conclusion: Fish collagen has good biocompatibility and wound repair ability. It is decomposed and utilized in the process of wound repair to form new tissues.

Optical coherence tomography for in vivo longitudinal monitoring of artificial dermal scaffold.

Artificial dermal scaffold (ADS) has undergone rapid development and been increasingly used for treating skin wound in clinics due to its good biocompatibility, controllable degradation, and low risk of disease infection. To obtain good treatment efficacy, ADS needs to be monitored longitudinally during the treatment process. For example, scaffold-tissue fit, cell in-growth, vascular regeneration, and scaffold degradation are the key properties to be inspected. However, to date, there are no effective, real-time, and noninvasive techniques to meet the requirement of the scaffold monitoring above. In this study, we propose to use optical coherence tomography (OCT) to monitor ADS in vivo through three-dimensional imaging. A swept source OCT system with a handheld probe was developed for in vivo skin imaging. Moreover, a cell in-growth, vascular regeneration, and scaffold degradation rate (IRDR) was defined with the volume reduction rate of the scaffold's collagen sponge layer. To measure the IRDR, a semiautomatic image segmentation algorithm was designed based on U-Net to segment the collagen sponge layer of the scaffold from OCT images. The results show that the scaffold-tissue fit can be clearly visualized under OCT imaging. The IRDR can be computed based on the volume of the segmented collagen sponge layer. It is observed that the IRDR appeared to a linear function of the time and in addition, the IRDR varied among different skin parts. Overall, it can be concluded that OCT has a good potential to monitor ADS in vivo. This can help guide the clinicians to control the treatment with ADS to improve the therapy.

Plug-and-Play Lymph Node-on-Chip: Secondary Tumor Modeling by the Combination of Cell Spheroid, Collagen Sponge and T-Cells

Towards the improvement of the efficient study of drugs and contrast agents, the 3D microfluidic platforms are currently being actively developed for testing these substances and particles in vitro. Here, we have elaborated a microfluidic lymph node-on-chip (LNOC) as a tissue engineered model of a secondary tumor in lymph node (LN) formed due to the metastasis process. The developed chip has a collagen sponge with a 3D spheroid of 4T1 cells located inside, simulating secondary tumor in the lymphoid tissue. This collagen sponge has a morphology and porosity comparable to that of a native human LN. To demonstrate the suitability of the obtained chip for pharmacological applications, we used it to evaluate the effect of contrast agent/drug carrier size, on the penetration and accumulation of particles in 3D spheroids modeling secondary tumor. For this, the 0.3, 0.5 and 4 μm bovine serum albumin (BSA)/tannic acid (TA) capsules were mixed with lymphocytes and pumped through the developed chip. The capsule penetration was examined by scanning with fluorescence microscopy followed by quantitative image analysis. The results show that capsules with a size of 0.3 μm passed more easily to the tumor spheroid and penetrated inside. We hope that the device will represent a reliable alternative to in vivo early secondary tumor models and decrease the amount of in vivo experiments in the frame of preclinical study.

A meta-analysis examined the effect of topical nursing application of antimicrobial as a prophylaxis for the stoppage of surgical wound infection in colorectal surgery.

To assess the impact of topical antimicrobial (TA) as a prophylaxis for the stoppage of surgical wound infection (SWI) in colorectal surgery (CS), we lead a meta-analysis. 9160 participants with CS were enrolled in the chosen studies; 4719 of them used TA, while 4441 served as controls. To assess the effectiveness of TA application in lowering SWIs following CS, odds ratios (OR) with 95% confidence intervals (CIs) were computed with a dichotomous technique with a fixed- or random-effect model. Significantly lower SWIs post CS for TA as whole (OR, 0.50; 95% CI, 0.38-0.64; P< .001), gentamicin collagen sponge and beads (OR, 0.52; 95% CI, 0.32-0.86; P= .01), triclosan impregnated fascial suture (OR, 0.57; 95% CI, 0.38-0.84; P= .005), antibiotic powder, ointment, lavage, or injection for the abdominal wound (OR, 0.35; 95% CI, 0.21-0.59; P< .001), and ionised silver dressing on the closed abdominal wound (OR, 0.45; 95% CI, 0.27-0.77; P= .003) compared to control. Significantly lower SWIs post CS for TA as a whole, gentamicin collagen sponge and beads, triclosan impregnated fascial sutures, antibiotic powder, ointment, lavage, or injection for the abdominal wound, and ionised silver dressing on the closed abdominal wound compared with control. The low sample size of 8 out of the 39 included studies in this meta-analysis calls for precaution when analysing the outcomes.

Chitosan/Hydroxyapatite Scaffolds with P28 as a Promising Osteoinductive Scaffold for Bone Healing Applications

Despite bone’s inherent ability to heal, large bone defects remain a major clinical concern. This study proposes an off-the-shelf treatment combining chitosan/hydroxyapatite (CS/HAp) scaffolds, covalently linked with either bone morphogenetic protein-2 (BMP-2) or its related peptide P28 via a UV crosslinking process. Although covalently binding the growth factors was reported as a great alternative to the conventionally physical adsorption and encapsulation methods, this method presents the risk of altering the molecular activity and interaction of the growth factors. Therefore, alkaline phosphatase (ALP) activity and alizarin red staining (ARS) with a quantitative cetylpyridinium chloride (CPC) assay were conducted to validate that our photo-crosslinking fabrication method did not interfere with the functionality of the growth factors. The ALP activity of C2C12 with 100 µg/mL P28 was found to be comparable to 0.5 µg/mL BMP-2 after two weeks, where 0.001 U/mL was recorded for both treatments. The C2C12 cultured with CS/HAp/BMP-2 and CS/HAp/P28 scaffolds also showed an increased ALP activity compared to the negative control. ARS-CPC assay presented the highest optical density in 0.3 µg/mL BMP-2 and 50 µg/mL P28, while the highest intensity of ARS was observed in C2C12 cultured with CS/HAp/BMP-2 and CS/HAp/P28 scaffolds compared to the negative controls. The osteoconductive capability of this delivery system was then investigated through a rat femoral condyle defect model, where the new bone mineral density and the bone volume increased for all CS/HAp scaffolds compared to the collagen sponge control treatment. The histological assessment showed a favourable bone regeneration efficacy of the CS/HAp/P28 compared to the CS/HAp/BMP-2 treatment, thus showing the use of CS/HAp scaffolds with P28 as a promising osteoinductive scaffold for bone healing applications.

Characterization of Dual-Layer Hybrid Biomatrix for Future Use in Cutaneous Wound Healing.

A skin wound without immediate treatment could delay wound healing and may lead to death after severe infection (sepsis). Any interruption or inappropriate normal wound healing, mainly in these wounds, commonly resulted in prolonged and excessive skin contraction. Contraction is a common mechanism in wound healing phases and contributes 40-80% of the original wound size post-healing. Even though it is essential to accelerate wound healing, it also simultaneously limits movement, mainly in the joint area. In the worst-case scenario, prolonged contraction could lead to disfigurement and loss of tissue function. This study aimed to fabricate and characterise the elastin-fortified gelatin/polyvinyl alcohol (PVA) film layered on top of a collagen sponge as a bilayer hybrid biomatrix. Briefly, the combination of halal-based gelatin (4% (w/v)) and PVA ((4% (w/v)) was used to fabricate composite film, followed by the integration of poultry elastin (0.25 mg/mL) and 0.1% (w/v) genipin crosslinking. Furthermore, further analysis was conducted on the composite bilayer biomatrix's physicochemical and mechanical strength. The bilayer biomatrix demonstrated a slow biodegradation rate (0.374967 ± 0.031 mg/h), adequate water absorption (1078.734 ± 42.33%), reasonable water vapour transmission rate (WVTR) (724.6467 ± 70.69 g/m2 h) and porous (102.5944 ± 28.21%). The bilayer biomatrix also exhibited an excellent crosslinking degree and was mechanically robust. Besides, the elastin releasing study presented an acceptable rate post-integration with hybrid biomatrix. Therefore, the ready-to-use bilayer biomatrix will benefit therapeutic effects as an alternative treatment for future diabetic skin wound management.

Wnt3a promotes odonto/osteogenic differentiation in vitro and tertiary dentin formation in a rat model.

To investigate the effect of Wnt3a on odonto/osteogenic differentiation of stem cells isolated from human exfoliated deciduous teeth (SHEDs) and reparative dentine formation in a rat model. Stem cells isolated from human exfoliated deciduous teeth were cultured in media with Wnt3a (50-200 ng/ml). Wnt activation was confirmed by β-catenin immunocytochemistry. Colony-forming unit assay (normalized percentage area), osteogenic gene expression analysis by real-time polymerase chain reaction and mineralization assays measured by the absorption at 540 nm were performed. Tertiary dentine formation in vivo was evaluated using 8-week-old, male Wistar rats. Cavities with pinpoint pulp exposure by a sharp instrument were prepared at the mesial surface of the first molars. Teeth were divided into (n=6): (1) distilled water (negative control), (2) phosphate-buffered saline (PBS), (3) lithium chloride in DI (20 μM), and (4) Wnt3a in PBS (200 ng/ml). Collagen sponge was used as a scaffold. The cavity was sealed with glass ionomer restoration. Four weeks later, animals were euthanized by sodium pentobarbital (120 mg/kg body weight). Hard tissue formation was evaluated using micro-computerized tomography. Sixty consecutive slides from the initial plane were analysed and calculated as bone/dentine volume per total tissue volume. Paraffin sections (2μm) were stained with haematoxylin and eosin and Masson's trichrome for morphological evaluation. Data are presented as the mean ± standard error. Mann-Whitney U test was used for two-group comparison. Kruskal Wallis followed by pairwise comparison was employed for three or more group comparisons. Statistical analysis was performed using GraphPad Prism 7. Differences were considered significant at p < .05. Wnt3a decreased SHEDs colony formation and increased OSX, BMP2, and DMP1 expression, corresponding to an increase in mineralization. Additionally, a significant increase in dentine/bone volume per total tissue volume was observed in Wnt3a treated defects. Dentine bridge formation at the exposure sites treated with Wnt3a demonstrated, while fibrous tissues were observed in the control. Wnt3a suppressed proliferation, increased osteogenic differentiation of SHEDs and promotes tertiary dentine formation. Wnt3a could be utilized as biological molecule for vital pulp therapy.

Can hydroxyapatite charged collagen sponge help reduce perioperative blood loss in adolescent idiopathic scoliosis surgery? Preliminary results in 68 patients.

Patient blood management has been recently emphasized to avoid perioperative blood transfusion in AIS surgery. Hydroxyapatite charged collagen sponge (HCS) is a bone substitute material made of collagen and ceramized hydroxyapatite, with associated haemostatic properties. The goal of this study was to assess the impact of HCS in the perioperative blood loss in AIS surgery. After IRB approval, all AIS patients undergoing primary correction were prospectively included over a 15-month period. Patients receiving HCS at the end of the procedure were compared to a control group (matched for age, gender, and fusion levels) without any haemostatic agent or bone substitute. The same perioperative blood saving strategies were used in both groups. Two subfascial drains were used for 48h in all patients. Perioperative blood loss and transfusion rates were analysed. A total of 34 patients were included in each group. No difference in drainage volume was observed at day 1, but the reduction was statistically different at day 3 (1135mL [800-1640] versus 930 [480-1510], p = 0.028, 0.63ml/Kg/h [0.4-0.92] versus 0.46 [0.29-0.7], p = 0.042). Multivariate analysis found that the use of HCS was associated with a decrease in the postoperative blood loss (OR = 1.17 [1.10-1.25]). The transfusion rate was lower in the HCS group [0 (0% vs. 3(8.8%), p = 0.076)]. No infection occurred, and no complication was reported. With 27% reduction in drain volume, hydroxyapatite charged collagen sponge can be considered as a blood salving strategy in AIS surgery. The role of the biomaterial in fusion rate still needs to be further assessed.

Evaluation of the Potential of Umbilical Cord Mesenchymal Stromal Cell–Derived Small Extracellular Vesicles to Improve Rotator Cuff Healing: A Pilot Ovine Study

Background: Despite significant advancements in surgical techniques to repair rotator cuff (RC) injuries, failure rates remain high and novel approaches to adequately overcome the natural biological limits of tendon and enthesis regeneration of the RC are required. Small extracellular vesicles (sEVs) derived from the secretome of human multipotent mesenchymal stromal cells (MSCs) have been demonstrated to modulate inflammation and reduce fibrotic adhesions, and therefore their local application could improve outcomes after RC repair. Purpose: In this pilot study, we evaluated the efficacy of clinical-grade human umbilical cord (hUC) MSC-derived sEVs (hUC-MSC-sEVs) loaded onto a type 1 collagen scaffold in an ovine model of acute infraspinatus tendon injury to improve RC healing. Study Design: Controlled laboratory study. Methods: sEVs were enriched from hUC-MSC culture media and were characterized by surface marker profiling. The immunomodulatory capacity was evaluated in vitro by T-cell proliferation assays, and particle count was determined by nanoparticle tracking analysis. Twelve skeletally mature sheep were subjected to partial infraspinatus tenotomy and enthesis debridement. The defects of 6 animals were treated with 2 × 1010 hUC-MSC-sEVs loaded onto a type 1 collagen sponge, whereas 6 animals received only a collagen sponge, serving as controls. Six weeks postoperatively, the healing of the infraspinatus tendon and the enthesis was evaluated by magnetic resonance imaging (MRI) and hard tissue histology. Results: CD3/CD28-stimulated T-cell proliferation was significantly inhibited by hUC-MSC-sEVs (P = .015) that displayed the typical surface marker profile, including the presence of the MSC marker proteins CD44 and melanoma-associated chondroitin sulfate proteoglycan. The local application of hUC-MSC-sEVs did not result in any marked systemic adverse events. Histologically, significantly improved Watkins scores (P = .031) indicated improved tendon and tendon-to-bone insertion repair after sEV treatment and lower postcontrast signal of the tendon and adjacent structures on MRI suggested less residual inflammation at the defect area. Furthermore, the formation of osteophytes at the injury site was significantly attenuated (P = .037). Conclusion: A local, single-dose application of hUC-MSC-sEVs promoted tendon and enthesis healing in an ovine model of acute RC injury. Clinical Relevance: Surgical repair of RC tears generally results in a clinical benefit for the patient; however, considerable rerupture rates have been reported. sEVs have potential as a cell-free biotherapeutic to improve healing outcomes after RC injury.

Local application of collagen sponge Alvokon after tooth extraction surgery

After tooth extraction, it is necessary to create favorable conditions for the healing of the hole of the removed tooth. Dynamic observation of the patient by the attending physician is generally accepted to assess the condition of the postoperative wound and the patient’s implementation of recommendations for oral care. However, patients often note the presence of pain and swelling of the mucous membrane of the well of the removed tooth.Stude – a comparative assessment of the course of the early postoperative period in patients with local application of collagen sponges Alvokon and Alvostaz.Materials and methods. A prospective randomized study of 80 patients aged 20 to 59 years was conducted. The patients underwent a tooth extraction operation under local anesthesia. The patients were divided into 4 groups of 20 people. In patients of the first group, a collagen sponge Alvocon No. 3 was placed in the hole of the removed tooth, in patients of the second group – a collagen sponge Alvocon No. 5 in patients of the third group – a hemostatic collagen sponge «Alvostaz». Local medicines were not used in patients of the fourth group after tooth extraction. In the postoperative period, the presence of pain and the condition of the hole of the removed tooth were assessed.Results. Wound healing proceeded by secondary tension without complications in all patients. Comparing the nature of the course of the early postoperative period, pain after tooth extraction in patients of different groups varied from 5 to 50% of cases, and the presence of edema of the wound edges – from 5 to 100% of cases.Conclusions. Pain in the early postoperative period is expressed 2 times less when using the collagen sponge Alvokon No. 5. Edema of the marginal gum of the well of the removed tooth was 1.1 times less when using the collagen sponge Alvokon, 1.2 times when using the hemostatic collagen sponge Alvostaz in comparison with the group of patients without the use of a sponge.

Evaluating the Degradation Process of Collagen Sponge and Acellular Matrix Implants In Vivo Using the Standardized HPLC-MS/MS Method

The purpose of this study was to establish a collagen determination method based on an isotope-labeled collagen peptide as an internal reference via high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS), and using the established method to evaluate the degradation process of collagen-based implants in vivo. The specific peptide (GPAGPQGPR) of bovine type I collagen was identified with an Orbitrap mass spectrometer. Then, the quantification method based on the peptide detection with HPLC-MS/MS was established and validated, and then further used to analyze the degradation trend of the collagen sponge and acellular matrix (ACM) in vivo at 2, 4, 6, 8, 12, 16, and 18 weeks after implantation. The results indicate that the relative standard deviation (RSD) of the detection precision and repeatability of the peptide-based HPLC-MS/MS quantification method were 3.55% and 0.63%, respectively. The limitations of quantification and detection were 2.05 × 10−3 μg/mL and 1.12 × 10−3 μg/mL, respectively. The collagen sponge and ACM were completely degraded at 10 weeks and 18 weeks, respectively. Conclusion: A specific peptide (GPAGPQGPR) of bovine type I collagen was identified with an Orbitrap mass spectrometer, and a standardized HPLC-MS/MS-based internal reference method for the quantification of bovine type I collagen was established. The method can be used for the analysis of the degradation of collagen-based implants in vivo.