Collagen matrix scaffolds:Future perspectives for the management of chronic liver diseases

Approximately 1.5 billion chronic liver disease(CLD)cases have been estimated worldwide,encompassing a wide range of liver damage severities.Moreover,liver disease causes approximately 1.75 million deaths per year.CLD is typically characterized by the silent and progressive deterioration of liver parenchyma due to an incessant inflammatory process,cell death,over deposition of extracellular matrix proteins,and dysregulated regeneration.Overall,these processes impair the correct function of this vital organ.Cirrhosis and liver cancer are the main complications of CLD,which accounts for 3.5%of all deaths worldwide.Liver transplantation is the optimal therapeutic option for advanced liver damage.The liver is one of the most common organs transplanted;however,only 10%of liver transplants are successful.In this context,regenerative medicine has made significant progress in the design of biomaterials,such as collagen matrix scaffolds,to address the limitations of organ transplantation(e.g.,low donation rates and biocompatibility).Thus,it remains crucial to continue with experimental and clinical studies to validate the use of collagen matrix scaffolds in liver disease.

Mineralized collagen scaffolds fabricated with amniotic membrane matrix increase osteogenesis under inflammatory conditions

Defects in craniofacial bones occur congenitally,after high-energy impacts,and during the course of treatment for stroke and cancer.These injuries are difficult to heal due to the overwhelming size of the injury area and the inflammatory environment surrounding the injury.Significant inflammatory response after injury may greatly inhibit regenerative healing.We have developed mineralized collagen scaffolds that can induce osteogenic differentiation and matrix biosynthesis in the absence of osteogenic media or supplemental proteins.The amniotic membrane is derived from placentas and has been recently investigated as an extracellular matrix to prevent chronic inflammation.Herein,we hypothesized that a mineralized collagen-amnion composite scaffold could increase osteogenic activity in the presence of inflammatory cytokines.We report mechanical properties of a mineralized collagen-amnion scaffold and investigated osteogenic differentiation and mineral deposition of porcine adipose-derived stem cells within these scaffolds as a function of inflammatory challenge.Incorporation of amniotic membrane matrix promotes osteogenesis similarly to un-modified mineralized collagen scaffolds,and increases in mineralized collagen-amnion scaffolds under inflammatory challenge.Together,these findings suggest that a mineralized collagen-amnion scaffold may provide a beneficial environment to aid craniomaxillofacial bone repair,especially in the course of defects presenting significant inflammatory complications.

Targeted protein delivery:carbodiimide crosslinking influences protein release from microparticles incorporated within collagen scaffolds

Tissue engineering response may be tailored via controlled,sustained release of active agents from protein-loaded degradable microparticles incorporated directly within three-dimensional(3D)ice-templated collagen scaffolds.However,the effects of covalent crosslinking during scaffold preparation on the availability and release of protein from the incorporated microparticles have not been explored.Here,we load 3D ice-templated collagen scaffolds with controlled additions of poly-(DL-lactide-co-glycolide)microparticles.We probe the effects of subsequent N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride crosslinking on protein release,using microparticles with different internal protein distributions.Fluorescein isothiocyanate labelled bovine serum albumin is used as a model protein drug.The scaffolds display a homogeneous microparticle distribution,and a reduction in pore size and percolation diameter with increased microparticle addition,although these values did not fall below those reported as necessary for cell invasion.The protein distribution within the microparticles,near the surface or more deeply located within the microparticles,was important in determining the release profile and effect of crosslinking,as the surface was affected by the carbodiimide crosslinking reaction applied to the scaffold.Crosslinking of microparticles with a high proportion of protein at the surface caused both a reduction and delay in protein release.Protein located within the bulk of the microparticles,was protected from the crosslinking reaction and no delay in the overall release profile was seen.

Injectable collagen scaffold with human umbilical cordderived mesenchymal stem cells promotes functional recovery in patients with spontaneous intracerebral hemorrhage:phase Ⅰ clinical trial

Animal expe riments have shown that injectable collagen scaffold with human umbilical cord-derived mesenchymal stem cells can promote recovery from spinal cord injury.To investigate whether injectable collagen scaffold with human umbilical cord-derived mesenchymal stem cells can be used to treat spontaneous intracerebral hemorrhage,this non-randomized phase I clinical trial recruited patients who met the inclusion criteria and did not meet the exclusion crite ria of spontaneous intracerebral hemorrhage treated in the Characteristic Medical Center of Chinese People’s Armed Police Force from May 2016 to December 2020.Patients were divided into three groups according to the clinical situation and patient benefit:control(n=18),human umbilical cord-derived mesenchymal stem cells(n=4),and combination(n=8).The control group did not receive any transplantation.The human umbilical cord-derived mesenchymal stem cells group received human umbilical cord-derived mesenchymal stem cell transplantation.The combination group received injectable collagen scaffold with human umbilical cord-derived mesenchymal stem cells.Patients who received injectable collagen scaffold with human umbilical cord-derived mesenchymal stem cells had more remarkable improvements in activities of daily living and cognitive function and smaller foci of intra cerebral hemorrhage-related encephalomalacia.Severe adve rse events associated with cell transplantation were not observed.Injectable collagen scaffold with human umbilical cord-derived mesenchymal stem cells appears to have great potential treating spontaneous intracerebral hemorrhage.

Collagen scaffold combined with human umbilical cord-mesenchymal stem cells transplantation for acute complete spinal cord injury

Currently, there is no effective strategy to promote functional recovery after a spinal cord injury. Collagen scaffolds can not only provide support and guidance for axonal regeneration, but can also serve as a bridge for nerve regeneration at the injury site. They can additionally be used as carriers to retain mesenchymal stem cells at the injury site to enhance their effectiveness. Hence, we hypothesized that transplanting human umbilical cord-mesenchymal stem cells on collagen scaffolds would enhance healing following acute complete spinal cord injury. Here, we test this hypothesis through animal studies and a phase I clinical trial.(1) Animal experiments: Models of completely transected spinal cord injury were established in rats and canines by microsurgery. Mesenchymal stem cells derived from neonatal umbilical cord tissue were adsorbed onto collagen scaffolds and surgically implanted at the injury site in rats and canines;the animals were observed after 1 week–6 months. The transplantation resulted in increased motor scores, enhanced amplitude and shortened latency of the motor evoked potential, and reduced injury area as measured by magnetic resonance imaging.(2) Phase I clinical trial: Forty patients with acute complete cervical injuries were enrolled at the Characteristic Medical Center of Chinese People's Armed Police Force and divided into two groups. The treatment group(n = 20) received collagen scaffolds loaded with mesenchymal stem cells derived from neonatal umbilical cordtissues;the control group(n = 20) did not receive the stem-cell loaded collagen implant. All patients were followed for 12 months. In the treatment group, the American Spinal Injury Association scores and activities of daily life scores were increased, bowel and urinary functions were recovered, and residual urine volume was reduced compared with the pre-treatment baseline. Furthermore, magnetic resonance imaging showed that new nerve fiber connections were formed, and diffusion tensor imaging showed that electrophysiological activity was recovered after the treatment. No serious complication was observed during follow-up. In contrast, the neurological functions of the patients in the control group were not improved over the follow-up period. The above data preliminarily demonstrate that the transplantation of human umbilical cord-mesenchymal stem cells on a collagen scaffold can promote the recovery of neurological function after acute spinal cord injury. In the future, these results need to be confirmed in a multicenter, randomized controlled clinical trial with a larger sample size. The clinical trial was approved by the Ethics Committee of the Characteristic Medical Center of Chinese People's Armed Police Force on February 3, 2016(approval No. PJHEC-2016-A8). All animal experiments were approved by the Ethics Committee of the Characteristic Medical Center of Chinese People's Armed Police Force on May 20, 2015(approval No. PJHEC-2015-D5).

Low-molecular-weight fucoidan inhibits the proliferation of melanoma via Bcl-2 phosphorylation and PTEN/AKT pathway

Fucoidan,a sulfate polysaccharide obtained from brown seaweed,has various bioactive properties,including anti-inflammatory,anti-cancer,anti-viral,anti-oxidant,anti-coagulant,anti-thrombotic,anti-angiogenic,and anti-Helicobacter pylori properties.However,the effects of low-molecular-weight fucoidan(LMW-F)on melanoma cell lines and three dimensional(3D)cell culture models are not well understood.This study aimed to investigate the effects of LMW-F on A375 human melanoma cells and cryopreserved biospecimens derived from patients with advanced melanoma.Ultrasonic wave was used to fragment fucoidan derived from Fucus vesiculosus into smaller LMW-F.MTT and live/dead assays showed that LMW-F inhibited cell proliferation in both A375 cells and patientderived melanoma explants in a 3D-printed collagen scaffold.The PTEN/AKT pathway was found to be involved in the anti-melanoma effects of fucoidan.Western blot analysis revealed that LMW-F reduced the phosphorylation of Bcl-2 at Thr 56,which was associated with the prevention of anti-apoptotic activity of cancer cells.Our findings suggested that LMW-F could enhance anti-melanoma chemotherapy and improve the outcomes of patients with melanoma resistance.

Collagen for brain repair:therapeutic perspectives

Biomaterials have increasingly become a focus of research on neuroprotection and neuroregeneration.Collagen,in terms of brain repair,presents many advantages such as being remarkably biocompatible,biodegradable,versatile and non-toxic.Collagen can be used to form injectable scaffolds and micro/nano spheres in order to：（i） locally release therapeutic factors with the aim of protecting degenerating neurons in neurodegenerative conditions such as Alzheimer＇s or Parkinson＇s diseases,（ii） encapsulate stem cells for safe delivery,（iii） encapsulate genetically modified cells to provide a long term source of trophic factors,（iv） fill in the voids from injury to serve as a structural support and provide a permissive microenvironment to promote axonal growth.This mini-review summarizes different applications of collagen biomaterial for central nervous system protection and repair,as well as the future perspectives.Overall,collagen is a promising natural biomaterial with various applications which has the potential to progress the development of therapeutic strategies in central nervous system injuries and degeneration.

Combination of mesenchymal stem cells and three-dimensional collagen scaffold preserves ventricular remodeling in rat myocardial infarction model

BACKGROUND Cardiovascular diseases are the major cause of mortality worldwide.Regeneration of the damaged myocardium remains a challenge due to mechanical constraints and limited healing ability of the adult heart tissue.Cardiac tissue engineering using biomaterial scaffolds combined with stem cells and bioactive molecules could be a highly promising approach for cardiac repair.Use of biomaterials can provide suitable microenvironment to the cells and can solve cell engraftment problems associated with cell transplantation alone.Mesenchymal stem cells(MSCs)are potential candidates in cardiac tissue engineering because of their multilineage differentiation potential and ease of isolation.Use of DNA methyl transferase inhibitor,such as zebularine,in combination with three-dimensional(3D)scaffold can promote efficient MSC differentiation into cardiac lineage,as epigenetic modifications play a fundamental role in determining cell fate and lineage specific gene expression.AIM To investigate the role of collagen scaffold and zebularine in the differentiation of rat bone marrow(BM)-MSCs and their subsequent in vivo effects.METHODS MSCs were isolated from rat BM and characterized morphologically,immunophenotypically and by multilineage differentiation potential.MSCs were seeded in collagen scaffold and treated with 3μmol/L zebularine in three different ways.Cytotoxicity analysis was done and cardiac differentiation was analyzed at the gene and protein levels.Treated and untreated MSC-seeded scaffolds were transplanted in the rat myocardial infarction(MI)model and cardiac function was assessed by echocardiography.Cell tracking was performed by DiI dye labeling,while regeneration and neovascularization were evaluated by histological and immunohistochemical analysis,respectively.RESULTS MSCs were successfully isolated and seeded in collagen scaffold.Cytotoxicity analysis revealed that zebularine was not cytotoxic in any of the treatment groups.Cardiac differentiation analysis showed more pronounced results in the type 3 treatment group which was subsequently chosen for the transplantation in the in vivo MI model.Significant improvement in cardiac function was observed in the zebularine treated MSC-seeded scaffold group as compared to the MI control.Histological analysis also showed reduction in fibrotic scar,improvement in left ventricular wall thickness and preservation of ventricular remodeling in the zebularine treated MSC-seeded scaffold group.Immunohistochemical analysis revealed significant expression of cardiac proteins in DiI labeled transplanted cells and a significant increase in the number of blood vessels in the zebularine treated MSC-seeded collagen scaffold transplanted group.CONCLUSION Combination of 3D collagen scaffold and zebularine treatment enhances cardiac differentiation potential of MSCs,improves cell engraftment at the infarcted region,reduces infarct size and improves cardiac function.

Study on the optical property and biocompatibility of a tissue engineering cornea

AIM: To study the optical property and biocompatibility of a tissue engineering cornea. METHODS: The cross-linker of N- (3-Dimethylaminoropyl)-N'ethylcarbodiimide hydrochloride (EDC)/ N-Hydroxysuccinimide (NHS) was mixed with Type I collagen at 10% (weight/volume). The final solution was molded to the shape of a corneal contact lens. The collagen concentrations of 10%, 12.5%, 15%, 17.5% and 20% artificial corneas were tested by UV/vis-spectroscopy for their transparency compared with normal rat cornea. 10-0 sutures were knotted on the edges of substitute to measure the corneal buttons's mechanical properties. Normal rat corneal tissue primary culture on the collagen scaffold was observed in 4 weeks. Histopathologic examinations were performed after 4 weeks of in vitro culturing. RESULTS: The collagen scaffold appearance was similar to that of soft contact lens. With the increase of collagen concentration, the transparency of artificial corneal buttons was diminished, but the toughness of the scaffold was enhanced. The scaffold transparency in the 10% concentration collagen group resembled normal rat cornea. To knot and embed the scaffold under the microscope, 20% concentration collagen group was more effective during implantation than lower concentrations of collagen group. In the first 3 weeks, corneal cell proliferation was highly active. The shapes of cells that grew on the substitute had no significant difference when compared with the cells before they were moved to the scaffold. However, on the fortieth day, most cells detached from the scaffold and died. Histopathologic examination of the primary culture scaffold revealed well grown corneal cells tightly attached to the scaffold in the former culturing. CONCLUSION: Collagen scaffold can be molded to the shape of soft contact corneal lens with NHS/EDC. The biological stability and biocompatibility of collagen from animal species may be used as material in preparing to engineer artificial corneal scaffold.

Embryoid bodies formation and differentiation from mouse embryonic stem cells in collagen/Matrigel scaffolds

Embryonic stem （ES） cells have the potential to develop into any type of tissue and are considered as a promising source of seeding cells for tissue engineering and transplantation therapy.The main catalyst for ES cells differentiation is the growth into embryoid bodies （EBs）,which are utilized widely as the trigger of in vitro differentiation.In this study,a novel method for generating EBs from mouse ES cells through culture in collagen/Matrigel scaffolds was successfully established.When single ES cells were seeded in three dimensional collagen/Matrigel scaffolds,they grew into aggregates gradually and formed simple EBs with circular structures.After 7 days＇ culture,they formed into cystic EBs that would eventually differentiate into the three embryonic germ layers.Evaluation of the EBs in terms of morphology and potential to differentiate indicated that they were typical in structure and could generate various cell types;they were also able to form into tissue-like structures.Moreover,with introduction of ascorbic acid,ES cells differentiated into cardiomyocytes efficiently and started contracting synchronously at day 19.The results demonstrated that collagen/Matrigel scaffolds supported EBs formation and their subsequent differentiation in a single three dimensional environment.

Silicified collagen scaffold induces semaphorin 3A secretion by sensory nerves to improve in-situ bone regeneration

Sensory nerves promote osteogenesis through the release of neuropeptides.However,the potential application and mechanism in which sensory nerves promote healing of bone defects in the presence of biomaterials remain elusive.The present study identified that new bone formation was more abundantly produced after implantation of silicified collagen scaffolds into defects created in the distal femur of rats.The wound sites were accompanied by extensive nerve innervation and angiogenesis.Sensory nerve dysfunction by capsaicin injection resulted in significant inhibition of silicon-induced osteogenesis in the aforementioned rodent model.Application of extracellular silicon in vitro induced axon outgrowth and increased expression of semaphorin 3 A(Sema3A)and semaphorin 4D(Sema4D)in the dorsal root ganglion(DRG),as detected by the upregulation of signaling molecules.Culture medium derived from silicon-stimulated DRG cells promoted proliferation and differentiation of bone marrow mesenchymal stem cells and endothelial progenitor cells.These effects were inhibited by the use of Sema3A neutralizing antibodies but not by Sema4D neutralizing antibodies.Knockdown of Sema3A in DRG blocked silicon-induced osteogenesis and angiogenesis almost completely in a femoral defect rat model,whereas overexpression of Sema3A promoted the silicon-induced phenomena.Activation of“mechanistic target of rapamycin”(mTOR)pathway and increase of Sema3A production were identified in the DRG of rats that were implanted with silicified collagen scaffolds.These findings support the role of silicon in inducing Sema3A production by sensory nerves,which,in turn,stimulates osteogenesis and angiogenesis.Taken together,silicon has therapeutic potential in orthopedic rehabilitation.

The Three-Dimensional Collagen Scaffold Improves the Sternness of Rat Bone Marrow Mesenchymal Stem Cells

Mesenchymal stem cells （MSCs） show the great promise for the treatment of a variety of diseases because of their self-renewal and multipotential abilities. MSCs are generally cultured on two-dimensional （2D） substrate in vitro. There are indications that they may simultaneously lose their sternness and multipotentiality as the result of prolonged 2D culture. In this study, we used three-dimensional （3D） collagen scaffolds as rat MSCs cartier and compared the properties of MSCs on 3D collagen scaffolds with monolayer cultured MSCs. The results demonstrated that collagen scaffolds were suitable for rat MSCs adherence and proliferation. More importantly, compared to MSCs under 2D culture, 3D MSCs significantly maintained higher expression levels of stemness genes （Oct4, Sox2, Rex-1 and Nanog）, yielded high frequencies of colony-forming units-fibroblastic （CFU-F） and showed enhanced osteogenic and adipogenic differentiation efficiency upon induction. Thus, 3D collagen scaffolds may be beneficial for expanding rat MSCs while maintaining the stem cell properties in vitro.

Injectable collagen scaffold promotes swine myocardial infarction recovery by long-term local retention of transplanted human umbilical cord mesenchymal stem cells

Stem cell therapy is an attractive approach for recovery from myocardial infarction(MI)but faces the challenges of rapid diffusion and poor survival after transplantation.Here we developed an injectable collagen scaffold to promote the long-term retention of transplanted cells in chronic MI.Forty-five minipigs underwent left anterior descending artery(LAD)ligation and were equally divided into three groups 2 months later(collagen scaffold loading with human umbilical mesenchymal stem cell(hUMSC)group,hUMSC group,and placebo group(only phosphate-buffered saline(PBS)injection)).Immunofluorescence staining indicated that the retention of transplanted cells was promoted by the collagen scaffold.Echocardiography and cardiac magnetic resonance imaging(CMR)showed much higher left ventricular ejection fraction(LVEF)and lower infarct size percentage in the collagen/hUMSC group than in the hUMSC and placebo groups at 12 months after treatment.There were also higher densities of vWf-,α-sma-,and cTnT-positive cells in the infarct border zone in the collagen/cell group,as revealed by immunohistochemical analysis,suggesting better angiogenesis and more cardiomyocyte survival after MI.Thus,the injectable collagen scaffold was safe and effective on a large animal myocardial model,which is beneficial for constructing a favorable microenvironment for applying stem cells in clinical MI.

Doping bioactive elements into a collagen scaffold based on synchronous self-assembly/mineralization for bone tissue engineering

Pure collagen is biocompatible but lacks inherent osteoinductive,osteoimmunomodulatory and antibacterial activities.To obtain collagen with these characteristics,we developed a novel methodology of doping bioactive elements into collagen through the synchronous self-assembly/mineralization(SSM)of collagen.In the SSM model,amorphous mineral nanoparticles(AMN)(amorphous SrCO3,amorphous Ag3PO4,etc.)stabilized by the polyampholyte,carboxymethyl chitosan(CMC),and collagen molecules were the primary components under acidic conditions.As the pH gradually increased,intrafibrillar mineralization occurred via the self-adaptive interaction between the AMNs and the collagen microfibrils,which were self-assembling;the AMNs wrapped around the microfibrils became situated in the gap zones of collagen and finally transformed into crystals.Srdoped collagen scaffolds(Sr-CS)promoted in vitro cell proliferation and osteogenic differentiation of rat bone marrow mesenchymal stromal cells(rBMSCs)and synergistically improved osteogenesis of rBMSCs by altering the macrophage response.Ag-doped collagen scaffolds(Ag-CS)exhibited in vitro antibacterial effects on S.aureus,as well as cell/tissue compatibility.Moreover,Sr-CS implanted into the calvarial defect of a rat resulted in improved bone regeneration.Therefore,the SSM model is a de novo synthetic strategy for doping bioactive elements into collagen,and can be used to fabricate multifunctional collagen scaffolds to meet the clinical challenges of encouraging osteogenesis,boosting the immune response and fighting severe infection in bone defects.

Development of an antimicrobial peptide-loaded mineralized collagen bone scaffold for infective bone defect repair

The repair of infective bone defects is a great challenge in clinical work.It is of vital importance to develop a kind of bone scaffold with good osteogenic properties and long-term antibacterial activity for local anti-infection and bone regeneration.A porous mineralized collagen(MC)scaffold containing poly(D,L-lactide-co-glycolic acid)(PLGA)microspheres loaded with two antibacterial synthetic peptides,Pac-525 or KSL-W was developed and characterized via scanning electron microscopy(SEM),porosity measurement,swelling and mechanical tests.The results showed that the MC scaffold embedded with smooth and compact PLGA microspheres had a positive effect on cell growth and also had antibacterial properties.Through toxicity analysis,cell morphology and proliferation analysis and alkaline phosphatase evaluation,the antibacterial scaffolds showed excellent biocompatibility and osteogenic activity.The antibacterial property evaluated with Staphylococcus aureus and Escherichia coli suggested that the sustained release of Pac-525 or KSL-W from the scaffolds could inhibit the bacterial growth aforementioned in the long term.Our results suggest that the antimicrobial peptides-loaded MC bone scaffold has good antibacterial and osteogenic activities,thus providing a great promise for the treatment of infective bone defects.

Transplantation of neural stem progenitor cells from different sources for severe spinal cord injury repair in rat

Neural stem progenitor cell(NSPC)transplantation has been regarded as a promising therapeutic method for spinal cord injury(SCI)repair.However,different NSPCs may have different therapeutic effects,and it is therefore important to identify the optimal NSPC type.In our study,we compared the transcriptomes of human fetal brain-derived NSPCs(BNSPCs),spinal cord-derived NSPCs(SCNSPCs)and H9 embryonic stem-cell derived NSPCs(H9-NSPCs)in vitro and subsequently we transplanted each NSPC type on a collagen scaffold into a T8-9 complete SCI rat model in vivo.In vitro data showed that SCNSPCs had more highly expressed genes involved in nerve-related functions than the other two cell types.In vivo,compared with BNSPCs and H9-NSPCs,SCNSPCs exhibited the best therapeutic effects;in fact,SCNSPCs facilitated electrophysiological and hindlimb functional recovery.This study demonstrates that SCNSPCs may be an appropriate candidate cell type for SCI repair,which is of great clinical significance.

Long-term clinical observation of patients with acute and chronic complete spinal cord injury after transplantation of Neuro Regen scaffold

Spinal cord injury(SCI)often results in an inhibitory environment at the injury site.In our previous studies,transplantation of a scaffold combined with stem cells was proven to induce neural regeneration in animal models of complete SCI.Based on these preclinical studies,collagen scaffolds loaded with the patients’own bone marrow mononuclear cells or human umbilical cord mesenchymal stem cells were transplanted into SCI patients.Fifteen patients with acute complete SCI and 51 patients with chronic complete SCI were enrolled and followed up for 2 to 5 years.No serious adverse events related to functional scaffold transplantation were observed.Among the patients with acute SCI,five patients achieved expansion of their sensory positions and six patients recovered sensation in the bowel or bladder.Additionally,four patients regained voluntary walking ability accompanied by reconnection of neural signal transduction.Among patients with chronic SCI,16 patients achieved expansion of their sensation level and 30 patients experienced enhanced reflexive defecation sensation or increased skin sweating below the injury site.Nearly half of the patients with chronic cervical SCI developed enhanced finger activity.These long-term follow-up results suggest that functional scaffold transplantation may represent a feasible treatment for patients with complete SCI.