Aptamer-Functionalized Bioscaffold Enhances Cartilage Repair by Improving Stem Cell Recruitment in Osteochondral Defects of Rabbit Knees

Background: Recruitment of endogenous stem cells has been considered an alternative to cell injection/implantation in articular cartilage repair. Purpose: (1) To develop a cartilage tissue-engineering scaffold with clinically available biomaterials and functionalize the scaffold with an aptamer (Apt19s) that specifically recognizes pluripotent stem cells. (2) To determine whether this scaffold could recruit joint-resident mesenchymal stem cells (MSCs) when implanted into an osteochondral defect in a rabbit model and to examine the effects of cartilage regeneration. Study Design: Controlled laboratory study. Methods: The reinforced scaffold was fabricated by embedding a silk fibroin sponge into silk fibroin/hyaluronic acid–tyramine hydrogel and characterized in vitro. A cylindrical osteochondral defect (3.2 mm wide × 4 mm deep) was created in the trochlear grooves of rabbit knees. The rabbits were randomly assigned into 3 groups: Apt19s-functionalized scaffold group, scaffold-only group, and control group. Animals were sacrificed at 6 and 12 weeks after transplantation. Repaired tissues were evaluated via gross examination, histologic examination, and immunohistochemistry. Results: In vitro, this aptamer-functionalized scaffold could recruit bone marrow–derived MSCs and support cell adhesion. In vivo, the aptamer-functionalized scaffold enhanced cell homing in comparison with the aptamer-free scaffold. The aptamer-functionalized scaffold group also exhibited superior cartilage restoration when compared with the scaffold-only group and the control group. Conclusion: The Apt19s-functionalized scaffold exhibited the ability to recruit MSCs both in vitro and in vivo and achieved a better outcome of cartilage repair than the scaffold only or control in an osteochondral defect model. Clinical Relevance: The findings demonstrate a promising strategy of using aptamer-functionalized bioscaffolds for restoration of chondral/osteochondral defects via aptamer-introduced homing of MSCs.

Download Full-text

A Novel Strategy to Enhance Microfracture Treatment With Stromal Cell-Derived Factor-1 in a Rat Model

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2020.595932 ◽

2021 ◽

Vol 8 ◽

Author(s):

Taylor Mustapich ◽

John Schwartz ◽

Pablo Palacios ◽

Haixiang Liang ◽

Nicholas Sgaglione ◽

...

Keyword(s):

Bone Marrow ◽

Cartilage Repair ◽

Osteochondral Defect ◽

Control Group ◽

Sprague Dawley ◽

Sprague Dawley Rats ◽

Stromal Cell Derived Factor ◽

Empty Control

BackgroundMicrofracture is one of the most widely used techniques for the repair of articular cartilage. However, microfracture often results in filling of the chondral defect with fibrocartilage, which exhibits poor durability and sub-optimal mechanical properties. Stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant for mesenchymal stem cells (MSCs) and is expressed at high levels in bone marrow adjacent to developing cartilage during endochondral bone formation. Integrating SDF-1 into an implantable collagen scaffold may provide a chondro-conductive and chondro-inductive milieu via chemotaxis of MSCs and promotion of chondrogenic differentiation, facilitating more robust hyaline cartilage formation following microfracture.ObjectiveThis work aimed to confirm the chemoattractive properties of SDF-1 in vitro and develop a one-step method for incorporating SDF-1 in vivo to enhance cartilage repair using a rat osteochondral defect model.MethodsBone marrow-derived MSCs (BMSCs) were harvested from the femurs of Sprague–Dawley rats and cultured in low-glucose Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, with the medium changed every 3 days. Passage 1 MSCs were analyzed by flow cytometry with an S3 Cell Sorter (Bio-Rad). In vitro cell migration assays were performed on MSCs by labeling cells with carboxyfluorescein diacetate, succinimidyl ester (CFDA-SE; Bio-Rad). For the microfracture model, a 1.6-mm-diameter osteochondral defect was created in the femoral trochleae of 20 Sprague–Dawley rats bilaterally until bone marrow spillage was seen under saline irrigation. One knee was chosen at random to receive implantation of the scaffold, and the contralateral knee was left unfilled as an empty control. Type I collagen scaffolds (Kensey Nash) were coated with either gelatin only or gelatin and SDF-1 using a dip coating process. The rats received implantation of either a gelatin-only scaffold (N = 10) or gelatin-and-SDF-1 scaffold (N = 10) at the site of the microfracture. Femurs were collected for histological analyses at 4- and 8-week time points post-operatively, and sections were stained with Safranin O/Fast Green. The samples were graded blindly by two observers using the Modified O’Driscoll score, a validated scoring system for chondral repair. A minimum of 10 separate grading scores were made per sample and averaged. Quantitative comparisons of cell migration in vitro were performed with one-way ANOVA. Cartilage repair in vivo was also compared among groups with one-way ANOVA, and the results were presented as mean ± standard deviation, with P-values < 0.05 considered as statistically significant.ResultsMSC migration showed a dose–response relationship with SDF-1, with an optimal dosage for chemotaxis between 10 and 100 ng/ml. After scaffold implantation, the SDF-1-treated group demonstrated complete filling of the cartilage defect with mature cartilage tissue, exhibiting strong proteoglycan content, smooth borders, and good incorporation into marginal cartilage. Modified O’Driscoll scores after 8 weeks showed a significant improvement of cartilage repair in the SDF-1 group relative to the empty control group (P < 0.01), with a trend toward improvement when compared with the gelatin-only-scaffold group (P < 0.1). No significant differences in scores were found between the empty defect group and gelatin-only group.ConclusionIn this study, we demonstrated a simple method for improving the quality of cartilage defect repair in a rat model of microfracture. We confirmed the chemotactic properties of SDF-1 on rat MSCs and found an optimized dosage range for chemotaxis between 10 and 100 ng/ml. Furthermore, we demonstrated a strategy to incorporate SDF-1 into gelatin–collagen I scaffolds in vivo at the site of an osteochondral defect. SDF-1-treated defects displayed robust hyaline cartilage resurfacing of the defect with minimal fibrous tissue, in contrast to the empty control group. The results of the in vitro and in vivo studies together suggest that SDF-1-mediated signaling may significantly improve the quality of cartilage regeneration in an osteochondral defect.

Download Full-text

Angiogenic property of silk fibroin scaffolds with adipose-derived stem cells on chick chorioallantoic membrane

Royal Society Open Science ◽

10.1098/rsos.201618 ◽

2021 ◽

Vol 8 (3) ◽

Cited By ~ 1

Author(s):

Tanapong Watchararot ◽

Weerapong Prasongchean ◽

Peerapat Thongnuek

Keyword(s):

Stem Cells ◽

Silk Fibroin ◽

Pore Diameter ◽

Chorioallantoic Membrane ◽

Control Group ◽

Adipose Derived Stem Cells ◽

Chick Chorioallantoic Membrane ◽

Human Adipose Stem Cells

Angiogenesis is a crucial step in tissue regeneration and repair. Biomaterials that allow or promote angiogenesis are thus beneficial. In this study, angiogenic properties of salt-leached silk fibroin (SF) scaffolds seeded with human adipose stem cells (hADSCs) were studied using chick chorioallantoic membrane (CAM) as a model. The hADSC-seeded SF scaffolds (SF-hADSC) with the porosity of 77.34 ± 6.96% and the pore diameter of 513.95 ± 4.99 µm were implanted on the CAM of chick embryos that were on an embryonic day 8 (E8) of development. The SF-hADSC scaffolds induced a spoke-wheel pattern of capillary network indicative of angiogenesis, which was evident since E11. Moreover, the ingrowth of blood vessels into the scaffolds was seen in histological sections. The unseeded scaffolds induced the same extent of angiogenesis later on E14. By contrast, the control group could not induce the same extent of angiogenesis. In vitro cytotoxicity tests and in vivo angioirritative study reaffirmed the biocompatibility of the scaffolds. This work highlighted that the biocompatible SF-hADSC scaffolds accelerate angiogenesis, and hence they can be a promising biomaterial for the regeneration of tissues that require angiogenesis.

Download Full-text

EFFECTS OF SHORT-TERM CYCLIC HYDROSTATIC PRESSURE ON INITIATING AND ENHANCING THE EXPRESSION OF CHONDROGENIC GENES IN HUMAN ADIPOSE-DERIVED MESENCHYMAL STEM CELLS

Journal of Mechanics in Medicine and Biology ◽

10.1142/s0219519414500547 ◽

2014 ◽

Vol 14 (04) ◽

pp. 1450054 ◽

Cited By ~ 9

Author(s):

FARZANEH SAFSHEKAN ◽

MOHAMMAD TAFAZZOLI SHADPOUR ◽

MOHAMMAD ALI SHOKRGOZAR ◽

NOOSHIN HAGHIGHIPOUR ◽

SEYED HAMED ALAVI

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Growth Factor ◽

Hydrostatic Pressure ◽

Chondrogenic Differentiation ◽

Negative Control ◽

Cartilage Tissue ◽

Control Group

Cartilage tissue engineering is a promising treatment for damaged or diseased cartilage that requires thorough understanding of influential parameters involved in chondrogenic differentiation. This study examined how 4-h application of cyclic hydrostatic pressure (CHP) of 5 MPa at 0.5 Hz could modulate chondroinduction of human adipose-derived mesenchymal stem cells (hAMSCs) in vitro. Four groups were examined including a negative control group, a chemical group treated by growth factor for 10 days, a mechanical group exposed to 4-h loading on the 10th day of pellet culture without any chondrogenic stimulator, and finally a chemical-mechanical group subjected to both growth factor and loading. Application of cyclic hydrostatic pressure increased the expression of chondrogenic genes, including sox9 and aggrecan to higher levels than those of the chemical group. This study indicates that cyclic hydrostatic pressure initiates and enhances the chondrogenic differentiation of mesenchymal stem cells with or without growth factors in vitro and confirms the important role of hydrostatic pressure during chondrogenesis in vivo.

Download Full-text

Nanopolymers Delivery of the Bone Morphogenetic Protein-4 Plasmid to Mesenchymal Stem Cells Promotes Articular Cartilage Repair In Vitro and In Vivo

Journal of Nanomaterials ◽

10.1155/2012/236953 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Junjun Shi ◽

Xin Zhang ◽

Yanbin Pi ◽

Jingxian Zhu ◽

Chunyan Zhou ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Articular Cartilage ◽

Cartilage Repair ◽

Viral Vectors ◽

Control Group ◽

Articular Cartilage Repair ◽

Experimental Group

The clinical application of viral vectors for gene therapy is limited for biosafety consideration. In this study, to promote articular cartilage repair, poly (lactic-co glycolic acid) (PLGA) nanopolymers were used as non-viral vectors to transfect rabbit mesenchymal stem cells (MSCs) with the pDC316-BMP4-EGFP plasmid. The cytotoxicity and transfection efficiency in vitro were acceptable measuring by CCK-8 and flow cytometry. After transfection, Chondrogenic markers (mRNA of Col2a1, Sox9, Bmp4, and Agg) of experimental cells (MSCs being transfected with BMP-4 plasmid by PLGA nanopolymers) were increased more than those of control cells (MSCs being transfected with naked BMP-4 plasmid alone). In vivo study, twelve rabbits (24 knees) with large full thickness articular cartilage defects were randomly divided into the experimental group (MSCs being transfected with BMP-4 plasmid by PLGA nanopolymers) and the control group (MSCs being transfected with naked BMP-4 plasmid). The experimental group showed better regeneration than the control group 6 and 12 weeks postoperatively. Hyaline-like cartilage formed at week 12 in the experimental group, indicating the local delivery of BMP-4 plasmid to MSCs by PLGA nanopolymers improved articular cartilage repair significantly. PLGA nanopolymers could be a promising and effective non-viral vector for gene therapy in cartilage repair.

Download Full-text

Characterisation of ovine bone marrow-derived stromal cells (oBMSC) and evaluation of chondrogenically induced micro-pellets for cartilage tissue repair in vivo

10.1101/2020.05.15.094847 ◽

2020 ◽

Cited By ~ 1

Author(s):

K. Futrega ◽

E. Music ◽

P.G. Robey ◽

S. Gronthos ◽

R.W. Crawford ◽

...

Keyword(s):

Bone Marrow ◽

Cartilage Repair ◽

Stromal Cells ◽

Articular Chondrocytes ◽

Cartilage Tissue ◽

Induction Medium ◽

Large Animal ◽

Osteochondral Defects

AbstractBackgroundBone marrow stromal cells (BMSC) show promise in cartilage repair, and sheep are the most common large animal pre-clinical model. The objective of this study was to characterize ovine BMSC (oBMSC) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSC or ovine articular chondrocytes (oACh) to repair osteochondral defects in sheep.MethodsoBMSC were characterised for surface marker expression using flow cytometry and evaluated for tri-lineage differentiation. oBMSC micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O2. The capacity of cartilage micro-pellets manufactured from oBMSC or oACh to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study. Expanded oBMSC were positive for CD44 and CD146 and negative for CD45.ResultsThe common adipogenic induction medium ingredient, 3-Isobutyl-1-methylxanthine (IBMX) was toxic to oBMSC, but adipogenesis could be restored by excluding IBMX from the medium. BMSC chondrogenesis was optimal in a 2% O2 atmosphere. Micro-pellets formed from oBMSC or oACh appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not.ConclusionThe sensitivity of oBMSC to IBMX highlights species-species differences between oBMSC and hBMSC. Micro-pellets manufactured from oBMSC were not effective in repairing osteochondral defects, while oACh micro-pellets enabled modest repair. While oBMSC can be driven to form cartilage-like tissue in vitro, their effective use in cartilage repair will require mitigation of hypertrophy.

Download Full-text

Mesenchymal Stem Cells Delivered in a Novel Cartilage Mimetic Hydrogel for the Treatment of Focal Chondral Lesions in an Equine Animal Model

Orthopaedic Journal of Sports Medicine ◽

10.1177/2325967119s00288 ◽

2019 ◽

Vol 7 (7_suppl5) ◽

pp. 2325967119S0028

Author(s):

Cecilia Pascual-Garrido ◽

Francisco Rodriguez-Fontan ◽

Masahiko Haneda ◽

Elizabeth Aisenbrey ◽

Karin Payne ◽

...

Keyword(s):

Stem Cells ◽

Cartilage Repair ◽

Poor Quality ◽

Quality Score ◽

Compressive Modulus ◽

Large Animal ◽

Osteochondral Defects

Objectives: A degradable biomaterial has been developed that resembles the native cartilage biochemical properties, in which stem cells can be seeded, differentiate and develop cartilaginous tissue. The purposes of this study were: 1) to determine if mesenchymal stem cells (MSCs) embedded in this cartilage mimetic hydrogel display in vitro chondrogenesis; 2) to demonstrate that the proposed hydrogel can be delivered in situ; and 3) to determine if the hydrogel ± MSCs supports in vivo chondrogenesis. Methods: A photopolymerizable hydrogel consisting of polyethylene glycol, CVPLSLYSGC, chondroitin sulfate (ChS), CRGDS and TGF-β3 was used. Equine bone marrow-derived MSCs were encapsulated in the hydrogel and cultured for 9 weeks. Compressive modulus was evaluated at day 1 and at weeks 3, 6 and 9. Chondrogenic differentiation was investigated via qPCR, Safranin-O staining and immunofluorescence. Three female horses were used. Two 15-mm width x 5-mm depth osteochondral defects were created bilaterally in the medial femoral condyle of each stifle joint. Five groups were established: Hydrogel (n=3), Hydrogel + MSCs (n=3), Microfracture (MFX, n=1), MFX + Hydrogel (n=3), and MFX + Hydrogel + MSCs (n=2). Repair tissue was evaluated at 6 months post intervention with the following cartilage repair scoring systems: macroscopically, International Cartilage Repair Society (ICRS); and histologically, the Modified O’Driscoll scoring (MODS) and ICRS II (Overall assessment 0%, fibrous -100%, hyaline cartilage).The ICRS parameter is scored using a 100-mm VAS, a score of 0 was assigned for properties considered indicative of poor quality and 100 for good quality. Results: In vitro, there was a significant increase in compressive modulus, collagen II and ChS as confirmation of chondrogenesis and hydrogel degradation. (Figure 1) In vivo, the hydrogel was readily photopolimerized in the defect. Cartilage repair was evident in all groups. As shown in Table 1, red indicates best quality score, blue means a poor quality score, but there was no statistical difference. According to the macroscopic ICRS, the hydrogel + MSCs performed better (P= 0.47). However, the MFX + Hydrogel + MSCs tended to perform better per the MODS (P= 0.61); and ICRS-Overall assessment (P= 0.9). Particularly, MFX showed the lowest score for subchondral bone(SCB) abnormalities (0% = abnormal, P= 0.09) but no inflammation was evident (100% = absent, P= 0.53), whereas the Hydrogel had the highest basal integration (100% = complete integration, P= 0.38) but presented moderate inflammation (Figure 2A). MFX showed SCB abnormalities and vascularization (Figure 2 B). Interestingly, a defect treated with MFX + Hydrogel presented more GAGs, less inflammation (vs Hydrogel) and less SCB abnormalities (vs MFX) (Figure 2C). Overall, the group performing better was MFX + Hydrogel + MSCs. Conclusion: This pilot study provides the first evidence of the ability to photopolymerize this novel hydrogel in situ and assess its ability to provide chondrogenic cues for cartilage repair in a large animal model. The presence of all three balanced factors (MFX, Hydrogel, MSCs) had higher scores per MODS summation and ICRS Overall assessment. Strengths of this study include: comparison of standard MFX therapy of osteochondral defects with a novel cartilage mimetic therapy; and use of a large animal that resembles the human knee biomechanically and anatomically. [Figure: see text][Figure: see text][Table: see text]

Download Full-text

Macroscopic and histologic evaluation of cartilage regeneration treated using xenogenic biodegradable porous sponge cartilage scaffold composite supplemented with allogenic adipose derived mesenchymal stem cells (ASCs) and secretome: An in vivo experimental study

Journal of Biomaterials Applications ◽

10.1177/0885328220934938 ◽

2020 ◽

Vol 35 (3) ◽

pp. 422-429

Author(s):

Lukas Widhiyanto ◽

Dwikora Novembri Utomo ◽

Adrianto Prasetyo Perbowo ◽

Kukuh Dwiputra Hernugrahanto ◽

Keyword(s):

Stem Cells ◽

Scoring System ◽

Zealand White Rabbit ◽

Cartilage Regeneration ◽

Cartilage Tissue ◽

Control Group ◽

Osteochondral Defects ◽

Femoral Trochlea ◽

Microscopic Evaluation

Introduction Articular cartilage is an avascular, alymphatic, and anisotropic tissue, these characteristics cause significant healing problems to injuries to the cartilage tissue. To overcome this problem, various techniques have been developed and widely used, but the cost-effectiveness and resulting tissue regeneration have never achieved hyaline-like cartilage that has the best biomechanical properties. The idea of this experiment is to use a Biodegradable Porous Sponge Cartilage (BPSC) Scaffold to enhance the regeneration of hyaline-like cartilage combined with microfracture technique and Adipose Derived Stem Cells (ASCs) or secretome on an animal model. Methods A model defect was made on the femoral trochlea of a New Zealand white rabbit. Four groups were made to compare different treatment methods for osteochondral defects. The groups were: (1) Control group; (2) Scaffold Group; (3) Scaffold + ASCs Group; (4) Scaffold + Secretome Group. After 12 weeks, we terminate the animal models, then a macroscopic evaluation using the International Cartilage Research Society (ICRS) scoring system and Oswestry Arthroscopy Score (OAS) was done, followed by sectioning the specimen for microscopic evaluation using the O’Driscoll scoring system. Results The mean score for all treatment group were better compared to the control group grossly and histologically. The best mean score for macroscopic and microscopic evaluation was the group given Scaffold + ASCs. Conclusion The application of BPSC scaffold enhances cartilage regeneration in larger osteochondral defects. Furthermore, the addition of ASCs or secretome along with the scaffold implantation further enhances the cartilage regeneration, in which ASCs shows better results.

Download Full-text

Characterisation of ovine bone marrow-derived stromal cells (oBMSC) and evaluation of chondrogenically induced micro-pellets for cartilage tissue repair in vivo

Stem Cell Research & Therapy ◽

10.1186/s13287-020-02045-3 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

K. Futrega ◽

E. Music ◽

P. G. Robey ◽

S. Gronthos ◽

R. Crawford ◽

...

Keyword(s):

Bone Marrow ◽

Cartilage Repair ◽

Stromal Cells ◽

Articular Chondrocytes ◽

Cartilage Tissue ◽

Large Animal ◽

Osteochondral Defects ◽

Tissue Integration

Abstract Bone marrow stromal cells (BMSC) show promise in cartilage repair, and sheep are the most common large animal pre-clinical model. Objective The objective of this study was to characterise ovine BMSC (oBMSC) in vitro, and to evaluate the capacity of chondrogenic micro-pellets manufactured from oBMSC or ovine articular chondrocytes (oACh) to repair osteochondral defects in sheep. Design oBMSC were characterised for surface marker expression using flow cytometry and evaluated for tri-lineage differentiation capacity. oBMSC micro-pellets were manufactured in a microwell platform, and chondrogenesis was compared at 2%, 5%, and 20% O2. The capacity of cartilage micro-pellets manufactured from oBMSC or oACh to repair osteochondral defects in adult sheep was evaluated in an 8-week pilot study. Results Expanded oBMSC were positive for CD44 and CD146 and negative for CD45. The common adipogenic induction ingredient, 3-Isobutyl-1-methylxanthine (IBMX), was toxic to oBMSC, but adipogenesis could be restored by excluding IBMX from the medium. BMSC chondrogenesis was optimal in a 2% O2 atmosphere. Micro-pellets formed from oBMSC or oACh appeared morphologically similar, but hypertrophic genes were elevated in oBMSC micro-pellets. While oACh micro-pellets formed cartilage-like repair tissue in sheep, oBMSC micro-pellets did not. Conclusion The sensitivity of oBMSC, compared to human BMSC, to IBMX in standard adipogenic assays highlights species-associated differences. Micro-pellets manufactured from oACh were more effective than micro-pellets manufactured from oBMSC in the repair of osteochondral defects in sheep. While oBMSC can be driven to form cartilage-like tissue in vitro, the effective use of these cells in cartilage repair will depend on the successful mitigation of hypertrophy and tissue integration.

Download Full-text