Preparation of collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold and its chondrogenic induction / 中国组织工程研究

BACKGROUND:Natural bone morphogenetic protein 2 disperses and degrades rapidly in vivo,reducing local concentration and therapeutic efficacy.Simply combining bone morphogenetic protein 2 with tissue engineering scaffolds could not stay in vivo for a long time,making it difficult to achieve good sustained and controlled release effects.OBJECTIVE:To prepare and test the biological properties and chondrogenic induction effect of collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold.METHODS:SD rat tail collagen was extracted and a collagen cartilage scaffold was prepared using a vacuum freeze-drying machine chemical crosslinking method.The plasmid expressing collagen-binding domain-bone morphogenetic protein 2 was constructed by rapid cloning C112 homologous recombination,constructed by genetic engineering,and introduced into E.coli,and then collagen-binding domain-bone morphogenetic protein 2 was isolated and purified.Natural bone morphogenetic protein 2 and collagen-binding domain-bone morphogenetic protein 2 were combined with collagen cartilage scaffolds,respectively,to detect the release level of bone morphogenetic protein 2 in the scaffolds.The biocompatibility of collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold was detected by CCK-8 assay and F-Actin staining.Bone marrow mesenchymal stem cells were implanted on two kinds of collagen cartilage scaffolds for chondrogenic induction,and their chondrogenic induction activity was tested.RESULTS AND CONCLUSION:(1)The binding rate of collagen-binding domain-bone morphogenetic protein 2 to collagen cartilage scaffolds was higher than that of natural bone morphogenetic protein 2(P<0.05).After being immersed in PBS for 7 days in vitro,the release of bone morphogenetic protein 2 in the collagen-binding domain bone morphogenetic protein 2-collagen cartilage scaffold was smaller than that in the natural bone morphogenetic protein 2-collagen cartilage scaffold(P<0.05).The results of the CCK-8 assay and F-Actin staining showed that the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold had no obvious cytotoxicity and had good biocompatibility.(2)After 14 days of chondrogenic induction,ELISA detection demonstrated that the expressions of agglutincan and type Ⅱ collagen A1 in the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold group were higher than those in the natural bone morphogenetic protein 2-collagen cartilage scaffold group(P<0.05).Under scanning electron microscopy,more bone marrow mesenchymal stem cells were observed on the inner wall of the pores of the two groups of scaffolds,and the cell morphology and size were the same,and the cells were closely arranged,without cell fragmentation or abnormal morphology.(3)The results indicate that the collagen-binding domain-bone morphogenetic protein 2-collagen cartilage scaffold has good biological properties and chondrogenic induction activity.

Targeting collagen in tumor extracellular matrix as a novel targeted strategy in cancer immunotherapy.

Collagen, the most abundant protein in mammal, is widely expressed in tissues and organs, as well as tumor extracellular matrix. Tumor collagen mainly accumulates in tumor stroma or beneath tumor blood vessel endothelium, and is exposed due to the fragmentary structure of tumor blood vessels. Through the blood vessels with enhanced permeability and retention (EPR) effect, collagen-binding macromolecules could easily bind to tumor collagen and accumulate within tumor, supporting tumor collagen to be a potential tumor-specific target. Recently, numerous studies have verified that targeting collagen within tumor extracellular matrix (TEM) would enhance the accumulation and retention of immunotherapy drugs at tumor, significantly improving their anti-tumor efficacy, as well as avoiding severe adverse effects. In this review, we would summarize the known collagen-binding domains (CBD) or proteins (CBP), their mechanism and application in tumor-targeting immunotherapy, and look forward to future development.

A collagen-binding SIRPαFc fusion protein for targeted cancer immunotherapy.

Collagen is abundant but exposed in tumor due to the abnormal tumor blood vessels, thus is considered as a tumor-specific target. The A3 domain of von Willebrand factor (vWF A3) is a kind of collagen-binding domain (CBD) which could bind collagen specifically. Previously we reported a chemosynthetic CBD-SIRPαFc conjugate, which could block CD47 and derived tumor-targeting ability by CBD. CBD-SIRPαFc conjugate represented improved anti-tumor efficacy with increased MHC II+ M1 macrophages, but the uncertain coupling ratio remained a problem. Herein, we produced a vWF A3-SIRPαFc fusion protein through eukaryotic expression system. It was examined at both molecular and cellular levels with its collagen affinity, uninfluenced original affinity to targets and phagocytosis-promoting function compared to unmodified SIRPαFc. Living imaging showed that vWF A3-SIRPαFc fusion protein derived the improved accumulation and retention in tumor than SIRPαFc. In the MC38 allograft model, vWF A3-SIRPαFc demonstrated a superior tumor-suppressing effect, characterized by increased MHC II+ M1 macrophages and T cells (particularly CD4+ T cells). These results revealed that vWF A3-SIRPαFc fusion protein derived tumor-targeting ability, leading to improved anti-tumor immunotherapeutic efficacy compared to SIRPαFc. Altogether, vWF A3 improved the anti-tumor efficacy and immune-activating function of SIRPαFc, supporting targeting tumor collagen as a possible targeted strategy.

Decellularized tendon scaffolds loaded with collagen targeted extracellular vesicles from tendon-derived stem cells facilitate tendon regeneration.

Stem cell-based treatment of tendon injuries remains to have some inherent issues. Extracellular vesicles derived from stem cells have shown promising achievements in tendon regeneration, though their retention in vivo is low. This study reports on the use of a collagen binding domain (CBD) to bind extracellular vesicles, obtained from tendon-derived stem cells (TDSCs), to collagen. CBD-extracellular vesicles (CBD-EVs) were coupled to decellularized bovine tendon sheets (DBTS) to fabricate a bio-functionalized scaffold (CBD-EVs-DBTS). Our results show that thus obtained bio-functionalized scaffolds facilitate the proliferation, migration and tenogenic differentiation of stem cells in vitro. Furthermore, the scaffolds promote endogenous stem cell recruitment to the defects, facilitate collagen deposition and improve the biomechanics of injured tendons, thus resulting in functional regeneration of tendons.

In silico design of fusion keratinocyte growth factor containing collagen-binding domain for tissue engineering application.

Keratinocyte growth factor (KGF) is a potential therapeutic factor in wound healing. However, its applications have been restricted due to its low stability, short half-life, and limited target specificity. We aimed to immobilize KGF on collagen-based biomaterials for long-lasting and targeted therapy by designing fusion forms of KGF with collagen-binding domains (CBD) from natural origins. Twelve fusion proteins were designed consisting of KGF and CBDs with different lengths and amino acid compositions. Three-dimensional (3D) structures of the fusions were predicted by homology modeling. Physiochemical properties and secondary structure of the fusions were evaluated by bioinformatics tools. Moreover, the effect of the CBDs on the 3D structure and dynamic behavior of the fusions was investigated by molecular dynamics (MD) simulation. The binding affinity of the fusions to collagen, KGF receptor, and heparin was assessed using docking tools. Our results demonstrated that fusions with small CBDs like CBD of mammalian collagenase and decapeptide CBD of von Willebrand factor (VWF) were more stable and properly folded than those with larger CBDs. On the other hand, the insertion of bulky CBDs, including Fibronectin CBD and CBD of Clostridium histolyticum collagenase, into KGF resulted in stronger binding to collagen. Therefore, very small or large CBDs are inappropriate for constructing KGF fusions because they suffer from low collagen affinity or poor stability. By comparing the results of MD simulation and docking, this study proposed that CBDs belonging to Vibrio mimicus metalloprotease and A3 domain of VWF would be good candidates to produce stable fusions with proper affinities toward collagen and KGF receptors. Moreover, the secondary structure analysis showed that the overall structure of KGF and CBDs was better preserved when CBDs were inserted at the C-terminal of KGF. This computational information about novel KGF fusions may help find the best constructs for experimental studies.

The advent of a pan-collagenous CLOVIS POINT for pathotropic targeting and cancer gene therapy, a retrospective.

The 'Clovis Point'-an enabling prehistoric gain-of-function in stone-age tool technologies which empowered the Paleoindian-Americans to hunt, to strike-deep, and to kill designated target megafauna more efficiently-was created biochemically by molecular-genetic bio-engineering. This Biomedical "Clovis Point" was crafted by adapting a broad-spectrum Pan-Collagen Binding Domain (Pan-Coll/CBD) found within the immature pre-pro-peptide segment of Von Willebrand Factor into a constructive series of advanced medical applications. Developed experimentally, preclinically, and clinically into a cutting-edge Biotechnology Platform, the Clovis Point is suitable for 1) solid-state binding of growth factors on collagenous scaffolds for improved orthopedic wound healing, 2) promoting regeneration of injured/diseased tissues; and 3) autologous stem cell capture, expansion, and gene-based therapies. Subsequent adaptations of the high-affinity Pan-Coll/CBD (exposed-collagen-seeking/surveillance function) for intravenous administration in humans, enabled the physiological delivery, aka Pathotropic Targeting to diseased tissues via the modified envelopes of gene vectors; enabling 4) precision tumor-targeting for cancer gene therapy and 5) adoptive/localized immunotherapies, demonstrating improved long-term survival value-thus pioneering a proximal and accessible cell cycle control point for cancer management-empowering modern medical oncologists to address persistent problems of chemotherapy resistance, recurrence, and occult progression of metastatic disease. Recent engineering adaptations have advanced the clinical utility to include the targeted delivery of small molecule APIs: including taxanes, mAbs, and RNA-based therapeutics.

Enhancement of keratinocyte growth factor potential in inducing adipose-derived stem cells differentiation into keratinocytes by collagen-targeting.

Different growth factors can regulate stem cell differentiation. We used keratinocyte growth factor (KGF) to direct adipose-derived stem cells (ASCs) differentiation into keratinocytes. To enhance KGF bioavailability, we targeted KGF for collagen by fusing it to collagen-binding domain from Vibrio mimicus metalloprotease (vibrioCBD-KGF). KGF and vibrioCBD-KGF were expressed in Escherichia coli and purified to homogeneity. Both proteins displayed comparable activities in stimulating proliferation of HEK-293 and MCF-7 cells. vibrioCBD-KGF demonstrated enhanced collagen-binding affinity in immunofluorescence and ELISA. KGF and vibrioCBD-KGF at different concentrations (2, 10, and 20 ng/ml) were applied for 21 days on ASCs cultured on collagen-coated plates. Keratinocyte differentiation was assessed based on morphological changes, the expression of keratinocyte markers (Keratin-10 and Involucrin), and stem cell markers (Collagen-I and Vimentin) by real-time PCR or immunofluorescence. Our results indicated that the expression of keratinocyte markers was substantially increased at all concentrations of vibrioCBD-KGF, while it was observed for KGF only at 20 ng/ml. Immunofluorescence staining approved this finding. Moreover, down-regulation of Collagen-I, an indicator of differentiation commitment, was more significant in samples treated with vibrioCBD-KGF. The present study showed that vibrioCBD-KGF is more potent in inducing the ASCs differentiation into keratinocytes compared to KGF. Our results have important implications for effective skin regeneration using collagen-based biomaterials.

A functionalized collagen-I scaffold delivers microRNA 21-loaded exosomes for spinal cord injury repair.

MicroRNA (miRNA)-based therapies have shown great potential in the repair of spinal cord injury (SCI). MicroRNA 21 (miR21) has been proven to have an essential protective effect on SCI. However, there are some challenges for miRNAs application due to their easy degradation and ineffective cell penetration. As natural vesicles, exosomes were considered ideal carriers for miRNAs delivery for their advantages of low immunogenicity, inherent stability and tissue/cell penetration. However, poor targeting and the low capacity of specific miRNAs impede their practical applications. This study aims to develop a type of genetically engineered miR21-loaded exosomes that can be entrapped in collagen-I (Col-I) scaffold to repair SCI. The collagen-binding domain (CBD)-fused lysosome-associated membrane glycoprotein 2b (Lamp2b) protein (CBD-LP) and miR21 were overexpressed in host HEK293T (293T) cells that were used to produce engineered miR21-loaded exosomes. The CBD peptide fused in Lamp2b on the exosome surface can stably tether exosomes to Col-I scaffold, facilitate the retention of miR21-loaded exosomes in lesion sites, promote the sustained release of miR21 to cells. Finally, a functionalized Col-I scaffold biomaterial enriched with miR21-loaded exosomes was developed and it could benefit the repair of SCI. STATEMENT OF SIGNIFICANCE: MiRNA-based therapeutics have promising potential in spinal cord injury (SCI) repair. However, easy degradation and ineffective cell penetration impede miRNAs application. Exosomes are natural vehicles for miRNAs delivery but face the challenge of diffusion in vivo. Here, the collagen-binding domain (CBD)-fused Lamp2b and miR21 were overexpressed in HEK293T cells to produce miR21-loaded and CBD-modified exosomes (CBD-LP-miR21-EXOs). The CBD modified on the exosome surface can stably tether exosomes to collagen-I scaffold to form functionalized CBD-LP-miR21-EXO-Col scaffold that can facilitate the retention of miR21-loaded exosomes, promote the sustained release of miR21 to cells and finally benefit SCI repair. Furthermore, this type of functionalized collagen-I materials can be widely applied for other tissue injury repairs by enriching the CBD-LP-EXOs loaded with appropriate miRNAs.

A Slotted Decellularized Osteochondral Scaffold With Layer-Specific Release of Stem Cell Differentiation Stimulators Enhances Cartilage and Bone Regeneration in Osteochondral Defects in a Rabbit Model.

BACKGROUND: Owing to the disappointing regenerative ability of osteochondral tissue, without treatment an osteochondral defect would progress to osteoarthritis. This situation motivates the need for new strategies to enhance the regeneration of osteochondral defects. PURPOSE: To develop a tissue-engineering scaffold by tethering bone morphogenetic protein 2 (BMP2) and transforming growth factor beta 3 (TGFß3) in a layer-specific manner on a slotted decellularized osteochondral matrix (SDOM) and to evaluate the efficacy of this scaffold for osteochondral regeneration. STUDY DESIGN: Controlled laboratory study. METHODS: Normal osteochondral tissue from the rabbit patellofemoral groove was sectioned into a slot shape and decellularized for fabricating an SDOM. The collagen-binding domain (CBD) was fused into the N-terminus of BMP2 or TGFß3 to synthesize 2 recombinant growth factors (GFs) (CBD-BMP2 or CBD-TGFß3), which were tethered to the bone layer and cartilage layer, respectively, of the SDOM to prepare a tissue-engineering scaffold (namely, CBD-GFs/SDOM). After examining the influence of the CBD-GFs/SDOM on the viability and layer-specific differentiation of bone marrow mesenchymal stem cells in vitro, we determined the regeneration potential of the CBD-GFs/SDOM on osteochondral regeneration in a rabbit model. A total of 72 New Zealand White rabbits with a cylindrical osteochondral defect in the patellofemoral groove were randomly assigned to 3 groups: defect only (control [CTL] group), defect patched with an SDOM (SDOM group), and defect patched with the CBD-GFs/SDOM (CBD-GFs/SDOM group). At 6 or 12 weeks postoperatively, the rabbits were euthanized to harvest the knee joint, which was then evaluated via gross observation, micro-computed tomography, histological staining, and mechanical testing. RESULTS: In vitro, the CBD-GFs/SDOM was noncytotoxic, showed high biomimetics with normal osteochondral tissue, was suitable for cell adhesion and growth, and had good layer-specific ability in inducing stem cell differentiation. Macroscopic images showed that the CBD-GFs/SDOM group had significantly better osteochondral regeneration than the CTL and SDOM groups had. Micro-computed tomography demonstrated that much more bony tissue was formed at the defect sites in the CBD-GFs/SDOM group compared with the defect sites in the CTL or SDOM group. Histological analysis showed that the CBD-GFs/SDOM group had a significant enhancement in osteochondral regeneration at 6 and 12 weeks postoperatively in comparison with the CTL or SDOM group. At 12 weeks postoperatively, the mechanical properties of reparative tissue were significantly better in the CBD-GFs/SDOM group than in the other groups. CONCLUSION: The CBD-GFs/SDOM is a promising scaffold for osteochondral regeneration. CLINICAL RELEVANCE: The findings of this study indicated that the CBD-GFs/SDOM is an excellent candidate for reconstructing osteochondral defects, which may be translated for clinical use in the future.

LL37 and collagen-binding domain-mediated LL37 binding with type I collagen: Quantification via QCM-D.

Antimicrobial peptide (AMP)-loaded biomaterials may represent a viable alternative for stimulating wound healing while protecting against infections. Previously, to develop an efficient delivery system for the cathelicidin antimicrobial peptide, LL37, our lab modified LL37 with a collagen-binding domain derived from collagenase (cCBD) as an anchoring unit to collagen-based wound dressings. However, a direct quantification of unmodified LL37 and cCBD-LL37 binding with collagen has not been performed. In this study, we used quartz crystal microbalance with dissipation monitoring (QCM-D), immunohistochemistry (IHC), and atomic force microscopy (AFM) to establish and characterize an adsorbed layer of type I collagen on the QCM-D sensor and quantify peptide-collagen binding. A collagen deposition protocol was successfully established by measuring concentration-dependent deposition of collagen in QCM-D, and collagen self-assembly was observed by IHC and AFM. Hydrophobicity is known to affect the behavior of collagen adsorption. Therefore, we compared the deposition of collagen on hydrophilic SiO2-coated sensors vs. hydrophobic polystyrene (PS)-coated sensors via QCM-D, and found that the hydrophobic surface yielded more collagen adsorption, which suggested that hydrophobic surfaces are preferable for collagen layer establishment. There was no significant difference between LL37 and cCBD-LL37 binding with collagen, but the cCBD-LL37 showed better retention on the collagen after washing with PBS, indicating that there is an advantage to using cCBD as an anchoring unit to collagen. Collectively, these results provide important information on cCBD-mediated AMP-binding mechanisms and establish an effective method for quantifying peptide-collagen binding.

Acellular dermal matrix decorated with collagen-affinity peptide accelerate diabetic wound healing through sustained releasing Histatin-1 mediated promotion of angiogenesis.

Treating diabetic ulcers is a major challenge in clinical practice, persecuting millions of patients with diabetes and increasing the medical burden. Recombinant growth factor application can accelerate diabetic wound healing via angiogenesis. The local administration of recombinant growth factors has no robust clinical efficiency because of the degradation of append short duration of the molecules in the hostile inflammatoryenvironment.The present study focused on the pathophysiology of impaired neovascularization and growth factor short duration in the diabetic wound. We prepared a collagen-binding domain (CBD)-fused recombinant peptide (C-Histatin-1) that had both pro-angiogenesis capacity and collagen-affinity properties. Next, we created a biocompatible acellular dermal matrix (ADM) as a drug delivery carrier that featured collagen-richness, high porosity, and non-cytotoxicity. C-Histatin-1 was then tethered on ADM to obtain a sustained-release effect. Finally, a functional scaffold (C-Hst1/ADM) was developed. C-Hst1/ADM can sustain-release Histatin-1 to promote the adhesion, migration, and angiogenesisof vascular endothelial cells in vitro. Using a diabetic wound model, we showed that C-Hst1/ADM could significantly promote angiogenesis, reduce scar widths, and improve extracellular collagen accumulation. Therefore, the results of this study provide a foundation for the clinical application of C-Hst1/ADM covering scaffold in the treatment of diabetic wounds.

Development of chimeric forms of the matrix metalloproteinase 2 collagen binding domain as artificial membrane binding proteins for targeting stem cells to cartilage lesions in osteoarthritic joints.

Targeting stem cells to cartilage lesions has the potential to enhance engraftment and chondrogenesis. Denatured type II collagen fibrils (gelatin) are exposed in lesions at the surface of osteoarthritic articular cartilage and are therefore ideal target sites. We have designed and investigated chimeric mutants of the three modules of the MMP-2 collagen binding domain (CBD) as potential ligands for stem cell targeting. We expressed full-length CBD for the first time and used it to identify the most important amino acid residues for binding to gelatin. Module 2 of CBD had the highest affinity binding to both Type I and Type II gelatin, whereas module 1 showed specificity for type II gelatin and module 3 for type I gelatin. We went on to generate chimeric forms of CBD consisting of three repeats of module 1 (111), module 2 (222) or module 3 (333). 111 lacked solubility and could not be further characterised. However 222 was found to bind to type II gelatin 14 times better than CBD, suggesting it would be optimal for attachment to cartilage lesions, whilst 333 was found to bind to type I gelatin 12 times better than CBD, suggesting it would be optimal for attachment to lesions in type I collagen-rich tissues. We coated 222 onto the external membrane of Mesenchymal Stem Cells and demonstrated higher attachment of the coated cells to type II gelatin than uncoated cells. We conclude that the three modules of CBD each have specific biological properties that can be exploited for targeting stem cells to cartilage lesions and other pathological sites.

The effect of poly(lactic-co-glycolic acid) conduit loading insulin-like growth factor 1 modified by a collagen-binding domain on peripheral nerve injury in rats.

Peripheral nerve injury (PNI) exists widely and seriously affects patients' daily lives. However, the effect of nerve repair is still limited, and only 50% of patients can recover useful functions. To overcome these obstacles, collagen-coated poly(lactic-co-glycolic acid) (PLGA) conduits loaded with CBD-IGF-1 were designed and tested in vitro and in vivo. The physical characterization of the conduit was tested by scanning electron microscopy, and the static water contact angle, release rate, and nerve regeneration ability of the conduit were verified in a rat sciatic nerve injury model. The results showed that the PLGA/col/CBD-IGF-1 conduit had a rough surface and good hydrophilicity. CBD-IGF-1 could be released slowly from the PLGA/col/CBD-IGF-1 conduit. In the in vivo experiment, gait analysis and electrophysiological evaluation showed that the sciatic functional index and electrophysiological parameters were best in the group treated with the PLGA/col/CBD-IGF-1 conduit. The pathological examination results for the sciatic nerve and gastrocnemius muscle in the group treated with the PLGA/col/CBD-IGF-1 conduit were better than those in the other three groups. In short, this study demonstrated the beneficial effects of CBD-IGF-1 in nerve regeneration. The PLGA/col/CBD-IGF-1 conduit has therapeutic potential for use in the treatment of PNI.

Mechanistic Insight into the Fragmentation of Type I Collagen Fibers into Peptides and Amino Acids by a Vibrio Collagenase.

Vibrio collagenases of the M9A subfamily are closely related to Vibrio pathogenesis for their role in collagen degradation during host invasion. Although some Vibrio collagenases have been characterized, the collagen degradation mechanism of Vibrio collagenase is still largely unknown. Here, an M9A collagenase, VP397, from marine Vibrio pomeroyi strain 12613 was characterized, and its fragmentation pattern on insoluble type I collagen fibers was studied. VP397 is a typical Vibrio collagenase composed of a catalytic module featuring a peptidase M9N domain and a peptidase M9 domain and two accessory bacterial prepeptidase C-terminal domains (PPC domains). It can hydrolyze various collagenous substrates, including fish collagen, mammalian collagens of types I to V, triple-helical peptide [(POG)10]3, gelatin, and 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-o-Arg (Pz-peptide). Atomic force microscopy (AFM) observation and biochemical analyses revealed that VP397 first assaults the C-telopeptide region to dismantle the compact structure of collagen and dissociate tropocollagen fragments, which are further digested into peptides and amino acids by VP397 mainly at the Y-Gly bonds in the repeating Gly-X-Y triplets. In addition, domain deletion mutagenesis showed that the catalytic module of VP397 alone is capable of hydrolyzing type I collagen fibers and that its C-terminal PPC2 domain functions as a collagen-binding domain during collagenolysis. Based on our results, a model for the collagenolytic mechanism of VP397 is proposed. This study sheds light on the mechanism of collagen degradation by Vibrio collagenase, offering a better understanding of the pathogenesis of Vibrio and helping in developing the potential applications of Vibrio collagenase in industrial and medical areas. IMPORTANCE Many Vibrio species are pathogens and cause serious diseases in humans and aquatic animals. The collagenases produced by pathogenic Vibrio species have been regarded as important virulence factors, which occasionally exhibit direct pathogenicity to the infected host or facilitate other toxins' diffusion through the digestion of host collagen. However, our knowledge concerning the collagen degradation mechanism of Vibrio collagenase is still limited. This study reveals the degradation strategy of Vibrio collagenase VP397 on type I collagen. VP397 binds on collagen fibrils via its C-terminal PPC2 domain, and its catalytic module first assaults the C-telopeptide region and then attacks the Y-Gly bonds in the dissociated tropocollagen fragments to release peptides and amino acids. This study offers new knowledge regarding the collagenolytic mechanism of Vibrio collagenase, which is helpful for better understanding the role of collagenase in Vibrio pathogenesis and for developing its industrial and medical applications.

Adenosine Deaminase 1 Overexpression Enhances the Antitumor Efficacy of Chimeric Antigen Receptor-Engineered T Cells.

Chimeric antigen receptor (CAR) T cell therapy mediates unprecedented benefit in certain leukemias and lymphomas, but has yet to achieve similar success in combating solid tumors. A substantial body of work indicates that the accumulation of adenosine in the solid tumor microenvironment (TME) plays a crucial role in abrogating immunotherapies. Adenosine deaminase 1 (ADA) catabolizes adenosine into inosine and is indispensable for a functional immune system. We have, for the first time, engineered CAR T cells to overexpress ADA. To potentially improve the pharmacokinetic profile of ADA, we have modified the overexpressed ADA in two ways, through the incorporation of a (1) albumin-binding domain or (2) collagen-binding domain. ADA and modified ADA were successfully expressed by CAR T cells and augmented CAR T cell exhaustion resistance. In a preclinical engineered ovarian carcinoma xenograft model, ADA and collagen-binding ADA overexpression significantly enhanced CAR T cell expansion, tumor tissue infiltration, tumor growth control, and overall survival, whereas albumin-binding ADA overexpression did not. Furthermore, in a syngeneic colon cancer solid tumor model, the overexpression of mouse ADA by cancer cells significantly reduced tumor burden and remodeled the TME to favor antitumor immunity. The overexpression of ADA for enhanced cell therapy is a safe, straightforward, reproducible genetic modification that can be utilized in current CAR T cell constructs to result in an armored CAR T product with superior therapeutic potential.

The Extracellular Matrix Enriched With Exosomes for the Treatment on Pulmonary Fibrosis in Mice.

Pulmonary fibrosis (PF) is a severe respiratory disease caused by lung microenvironment changes. TGF-ß/Smad3 signaling pathway plays a critical role in the fibrotic process. MicroRNA-29 (miR-29) has proved to alleviate the occurrence of PF by downregulating TGF-ß/Smad3 signaling pathway. The miRNA application encounters obstacles due to its low stability in body and no targeting to lesions. Exosomes can be used for therapeutic delivery of miRNA due to their favorable delivery properties. However, low efficiency of separation and production impedes the therapeutic application of exosomes. In this study, we developed a liquid natural extracellular matrix (ECM) enriched with miR-29-loaded exosomes for PF treatment. The collagen-binding domain (CBD)-fused Lamp2b (CBD-Lamp2b) and miR-29 were overexpressed in human foreskin fibroblast (HFF) host cells for the entrapment of miR-29-loaded exosomes in ECM of the cells. The repeated freeze-thaw method was performed to prepare the liquid ECM enriched with exosomes without destroying the exosomal membrane. In summary, this study developed a novel functional ECM biomaterial for therapy of PF, and also provided a promising gene therapy platform for different diseases by treatment with liquid ECM that is, enriched with exosomes loaded with different functional miRNAs.

Cell-derived extracellular matrix enhanced by collagen-binding domain-decorated exosomes to promote neural stem cells neurogenesis.

Enhancing neurogenesis of neural stem cells (NSCs) is crucial in stem cell therapy for neurodegenerative diseases. Within the extracellular microenvironment, extracellular matrix (ECM) plays a pivotal role in modulating cell behaviors. However, a single ECM biomaterial is not sufficient to establish an ideal microenvironment. As multifunctional nanocarriers, exosomes display tremendous advantages for the treatments of various diseases. Herein, collagen binding domain peptide-modified exosomes (CBD-Exo) were obtained from the SH-SY5Y cell line infected with lentivirus particles encoding CBD-lysosome associated membrane glycoprotein 2b (CBD-Lamp2b) to improve the binding efficiency of exosomes and ECM. An exosomes-functionalized ECM (CBD-Exo/ECM) was then constructed via the interaction between CBD and collagen in ECM. Then, CBD-Exo/ECM was employed as a carrier for NSCs culture. The results showed that CBD-Exo/ECM can support the neurogenesis of NSCs with the percentage of proliferation marker EdU-positive (35.8% ± 0.47% vs 21.9% ± 2.32%) and neuron maker Tuj-1-positive (55.8% ± 0.47% vs 30.6% ± 2.62%) were both significantly increased in the exosomes-functionalized ECM system. This exosomes-functionalized ECM was capable to promote the cell proliferation and accelerate neuronal differentiation of NSCs, providing a potential biomedical material for stem cell application in tissue engineering and regenerative medicine.

Adipose-Derived Stromal Cell-Sheets Sandwiched, Book-Shaped Acellular Dermal Matrix Capable of Sustained Release of Basic Fibroblast Growth Factor Promote Diabetic Wound Healing.

The management of diabetic wounds is a therapeutic challenge in clinical settings. Current tissue engineering strategies for diabetic wound healing are insufficient, owing to the lack of an appropriate scaffold that can load a large number of stem cells and induce the interaction of stem cells to form granulation tissue. Herein we fabricated a book-shaped decellularized dermal matrix (BDDM), which shows a high resemblance to native dermal tissue in terms of its histology, microstructure, and ingredients, is non-cytotoxic and low-immunogenic, and allows adipose-derived stromal cell (ASC) attachment and proliferation. Then, a collagen-binding domain (CBD) capable of binding collagen was fused into basic fibroblast growth factor (bFGF) to synthetize a recombinant growth factor (termed as CBD-bFGF). After that, CBD-bFGF was tethered onto the collagen fibers of BDDM to improve its endothelial inducibility. Finally, a functional scaffold (CBD-bFGF/BDDM) was fabricated. In vitro and in vivo experiments demonstrated that CBD-bFGF/BDDM can release tethered bFGF with a sustained release profile, steadily inducing the interaction of stem cells down to endothelial differentiation. ASCs were cultured to form a cell sheet and then sandwiched by CBD-bFGF/BDDM, thus enlarging the number of stem cells loaded into the scaffold. Using a rat model, the ASC sheets sandwiched with CBD-bFGF/BDDM (ASCs/CBD-bFGF/BDDM) were capable of enhancing the formation of granulation tissue, promoting angiogenesis, and facilitating collagen deposition and remodeling. Therefore, the findings of this study demonstrate that ASCs/CBD-bFGF/BDDM could be applicable for diabetic wound healing.

Cell-Free Book-Shaped Decellularized Tendon Matrix Graft Capable of Controlled Release of BMP-12 to Improve Tendon Healing in a Rat Model.

BACKGROUND: Achilles tendon (AT) defects often occur in traumatic and chronic injuries. Currently, no graft can satisfactorily regenerate parallel tendinous tissue at the defect site to completely restore AT function. PURPOSE: To develop a cell-free functional graft by tethering bone morphogenetic protein 12 (BMP-12) on a book-shaped decellularized tendon matrix (BDTM) and to determine whether this graft is more beneficial for AT defect healing than an autograft. STUDY DESIGN: Controlled laboratory study. METHODS: Canine patellar tendon was sectioned into a book shape and decellularized to fabricate a BDTM. The collagen-binding domain (CBD) was fused into the N-terminus of BMP-12 to synthesize a recombinant BMP-12 (CBD-BMP-12), which was tethered to the BDTM to prepare a cell-free functional graft (CBD-BMP-12/BDTM). After its tensile resistance, tenogenic inducibility, and BMP-12 release dynamics were evaluated, the efficacy of the graft for tendon regeneration was determined in a rat model. A total of 140 mature male Sprague-Dawley rats underwent AT tenotomy. The defect was reconstructed with reversed AT (autograft group), native BMP-12 tethered to an intact decellularized tendon matrix (IDTM; NAT-BMP-12/IDTM group), native BMP-12 tethered to a BDTM (NAT-BMP-12/BDTM group), CBD-BMP-12 tethered on an IDTM (CBD-BMP-12/IDTM group), and CBD-BMP-12 tethered on a BDTM (CBD-BMP-12/BDTM group). The rats were sacrificed 4 or 8 weeks after surgery to harvest AT specimens. Six specimens from each group at each time point were used for histological evaluation; the remaining 8 specimens were used for biomechanical testing. RESULTS: In vitro CBD-BMP-12/BDTM was noncytotoxic, showed high biomimetics with native tendons, was suitable for cell adhesion and growth, and had superior tenogenic inducibility. In vivo the defective AT in the CBD-BMP-12/BDTM group regenerated more naturally than in the other groups, as indicated by more spindle-shaped fibroblasts embedded in a matrix of parallel fibers. The biomechanical properties of the regenerated AT in the CBD-BMP-12/BDTM group also increased more significantly than in the other groups. CONCLUSION: CBD-BMP-12/BDTM is more beneficial than autograft for healing AT defects in a rat model. CLINICAL RELEVANCE: The findings of this study demonstrate that CBD-BMP-12/BDTM can serve as a practical graft for reconstructing AT defects.

Brain-derived neurotrophic factor fused with a collagen-binding domain inhibits neuroinflammation and promotes neurological recovery of traumatic brain injury mice via TrkB signalling.

OBJECTIVES: As one of the vital nutrient factors in central nervous system (CNS), brain-derived neurotrophic factor (BDNF) can significantly attenuate neuron damage and promote neurogenesis. Nevertheless, little research has been conducted on regulating the effect of BDNF on the inflammatory response after traumatic brain injury (TBI). METHODS: In this study, we used BDNF fused with a collagen-binding domain (CBD-BDNF) to maintain a sufficient concentration of BDNF in the TBI hemisphere, and then, the regulatory effects of BDNF and CBD-BDNF on the inflammatory response of microglia were investigated both on a TBI mice model in vivo and LPS-stimulated microglia experiment in vitro. KEY FINDINGS: The results revealed that BDNF and CBD-BDNF had similar effects on attenuating the pro-inflammatory reactions but promoting anti-inflammatory responses of microglia induced by LPS in vitro. Furthermore, CBD-BDNF significantly improved the neurological behaviours of TBI mice and alleviated the inflammatory reaction after TBI, while BDNF had weaker effects compared with those of CBD-BDNF. Additionally, the TrkB inhibitor K252a significantly inhibited the above effects of CBD-BDNF. CONCLUSIONS: In conclusion, CBD-BDNF can promote the anti-inflammatory function of microglia and neurological recovery of TBI mice through TrkB signalling.