An In Vitro System to Study the Effect of Subchondral Bone Health on Articular Cartilage Repair in Humans

Chondrocyte-based cartilage repair strategies, such as articular chondrocyte implantation, are widely used, but few studies addressed the communication between native subchondral bone cells and the transplanted chondrocytes. An indirect co-culture model was developed, representing a chondrocyte/scaffold-construct repair of a cartilage defect adjoining bone, where the bone could have varying degrees of degeneration. Human BM-MSCs were isolated from two areas of subchondral bone in each of five osteochondral tissue specimens from five patients undergoing knee arthroplasty. These two areas underlaid the macroscopically and histologically best and worst cartilage, representing early and late-stage OA, respectively. BM-MSCs were co-cultured with normal chondrocytes suspended in agarose, with the two cell types separated by a porous membrane. After 0, 7, 14 and 21 days, chondrocyte–agarose scaffolds were assessed by gene expression and biochemical analyses, and the abundance of selected proteins in conditioned media was assessed by ELISA. Co-culture with late-OA BM-MSCs resulted in a reduction in GAG deposition and a decreased expression of genes encoding matrix-specific proteins (COL2A1 and ACAN), compared to culturing with early OA BM-MSCs. The concentration of TGF-β1 was significantly higher in the early OA conditioned media. The results of this study have clinical implications for cartilage repair, suggesting that the health of the subchondral bone may influence the outcomes of chondrocyte-based repair strategies.

Download Full-text

Microvalve bioprinting of MSC-Chondrocyte Co-Cultures

10.20944/preprints202109.0357.v1 ◽

2021 ◽

Author(s):

Joseph Dudman ◽

Ana Marina Ferreira ◽

Piergiorgio Gentile ◽

Xiao Wang ◽

Kenneth Dalgarno

Keyword(s):

High Throughput Screening ◽

Cellular Therapy ◽

Cell Types ◽

Inhibitory Effect ◽

Proof Of Concept ◽

Concept Study ◽

Indirect Immunofluorescence Staining ◽

Chondrocyte Implantation ◽

Tissue Models

Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof of concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures as an in vitro model of autologous chondrocyte implantation (ACI), an established cellular therapy for osteoarthritis. Microvalve-based bioprinting uses microvalves to deposit cells suspended in a liquid in a consistent and repeatable manner. In this case MSCs and chondrocytes have been sequentially deposited into an insert based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate improved repair. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This proof-of-concept study therefore provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.

Download Full-text

Evolutionary insights into primate skeletal gene regulation using a comparative cell culture model

10.1101/2021.09.30.462680 ◽

2021 ◽

Author(s):

Genevieve Housman ◽

Emilie Briscoe ◽

Yoav Gilad

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Bone Cells ◽

Cell Types ◽

Differentially Expressed ◽

Functional Genomic ◽

Cell Culture Model ◽

Culture Model ◽

Study Gene Regulation

AbstractThe evolution of complex skeletal traits in primates was likely influenced by both genetic and environmental factors. Because skeletal tissues are notoriously challenging to study using functional genomic approaches, they remain poorly characterized even in humans, let alone across multiple species. The challenges involved in obtaining functional genomic data from the skeleton, combined with the difficulty of obtaining such tissues from nonhuman apes, motivated us to consider an alternative in vitro system with which to comparatively study gene regulation in skeletal cell types. Specifically, we differentiated six human and six chimpanzee induced pluripotent stem cell lines (iPSCs) into mesenchymal stem cells (MSCs) and subsequently into osteogenic cells (bone cells). We validated differentiation using standard methods and collected single-cell RNA sequencing data from over 100,000 cells across multiple samples and replicates at each stage of differentiation. While most genes that we examined display conserved patterns of expression across species, hundreds of genes are differentially expressed (DE) between humans and chimpanzees within and across stages of osteogenic differentiation. Some of these interspecific DE genes show functional enrichments relevant in skeletal tissue trait development. Moreover, topic modeling indicates that interspecific gene programs become more pronounced as cells mature. Overall, we propose that this in vitro model can be used to identify interspecific regulatory differences that may have contributed to skeletal trait differences between species.Author SummaryPrimates display a range of skeletal morphologies and susceptibilities to skeletal diseases, but the molecular basis of these phenotypic differences is unclear. Studies of gene expression variation in primate skeletal tissues are extremely restricted due to the ethical and practical challenges associated with collecting samples. Nevertheless, the ability to study gene regulation in primate skeletal tissues is crucial for understanding how the primate skeleton has evolved. We therefore developed a comparative primate skeletal cell culture model that allows us to access a spectrum of human and chimpanzee cell types as they differentiate from stem cells into bone cells. While most gene expression patterns are conserved across species, we also identified hundreds of differentially expressed genes between humans and chimpanzees within and across stages of differentiation. We also classified cells by osteogenic stage and identified additional interspecific differentially expressed genes which may contribute to skeletal trait differences. We anticipate that this model will be extremely useful for exploring questions related to gene regulation variation in primate bone biology and development.

Download Full-text

Bone Marrow Progenitor Cells Isolated from Young Rabbit Trochlea Are More Numerous and Exhibit Greater Clonogenic, Chondrogenic, and Osteogenic Potential than Cells Isolated from Condyles

Cartilage ◽

10.1177/1947603517693044 ◽

2017 ◽

Vol 9 (4) ◽

pp. 378-390 ◽

Cited By ~ 3

Author(s):

Garima Dwivedi ◽

Anik Chevrier ◽

Caroline D. Hoemann ◽

Michael D. Buschmann

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Cartilage Repair ◽

Subchondral Bone ◽

Biological Properties ◽

Biological Characteristics ◽

Cell Yield ◽

Young Rabbit ◽

Bone Marrow Progenitor

Objective. Bone marrow stimulation procedures initiate repair by fracturing or drilling subchondral bone at base of cartilaginous defect. Earlier studies have shown that defect location and animal age affect cartilage repair outcome, suggesting a strong influence of structural and biological characteristics of subchondral bone. Here, we analyzed comprehensive biological characteristics of bone marrow progenitor cells (BMPCs) in subchondral bone of young and old rabbit condyle and trochlea. We tested the hypothesis that in vitro biological properties of BMPCs are influenced by location, age of donor and method of their isolation. Design. In vitro biological properties, including cell yield, colony-forming unit fibroblasts (CFU-f), surface marker expression, and differentiation potential were determined. Comparisons were carried out between trochlea versus condyle and epiphyseal versus metaphyseal bone using old ( N = 5) and young animal knees ( N = 8) to generate collagenase and explant-derived BMPC cultures. Results. CFU-f, cell yield, expression of stem cell markers, and osteogenic differentiation were significantly superior for younger animals. Trochlear subchondral bone yielded the most progenitors with the highest clonogenic potential and cartilaginous matrix expression. Trochlear collagenase-derived BMPCs had higher clonogenic capacity than explant-derived ones. Epiphyseal cells generated a larger chondrogenic pellet mass than metaphyseal-derived BMPCs. All older pellet cultures and one non-responder young rabbit failed to accumulate glycosaminoglycans (GAGs). Conclusion. Taken together, these results suggest that properties intrinsic to subchondral progenitors could significantly influence cartilage repair potential, and could partly explain variability in cartilage repair outcomes using same cartilage repair approach.

Download Full-text

Optimization of Degradation Profile for New Scaffold in Cartilage Repair

Cartilage ◽

10.1177/1947603517700954 ◽

2017 ◽

Vol 9 (4) ◽

pp. 438-449 ◽

Cited By ~ 8

Author(s):

Sarav S. Shah ◽

Haixiang Liang ◽

Sandeep Pandit ◽

Zalak Parikh ◽

John A. Schwartz ◽

...

Keyword(s):

Cartilage Repair ◽

Subchondral Bone ◽

Rat Model ◽

Articular Chondrocytes ◽

Short Term ◽

Morphological Studies ◽

Biomaterial Scaffold

Objective To establish whether a novel biomaterial scaffold with tunable degradation profile will aid in cartilage repair of chondral defects versus microfracture alone in vitro and in a rat model in vivo. Design In vitro—Short- and long-term degradation scaffolds were seeded with culture expanded articular chondrocytes or bone marrow mesenchymal stem cells. Cell growth and differentiation were evaluated with cell morphological studies and gene expression studies. In vivo—A microfracture rat model was used in this study to evaluate the repair of cartilage and subchondral bone with the contralateral knee serving as the empty control. The treatment groups include (1) empty osteochondral defect, (2) polycaprolactone copolymer–based polyester polyurethane–urea (PSPU-U) caffold short-term degradative profile, and (3) PSPU-U scaffold long-term degradative profile. After placement of the scaffold, the rats were then allowed unrestricted activity as tolerated, and histological analyses were performed at 4, 8, and 16 weeks. The cartilage defect was measured and compared with the contralateral control side. Results In vitro—Long-term scaffolds showed statistically significant higher levels of aggrecan and type II collagen expression compared with short-term scaffolds. In vivo—Within 16 weeks postimplantation, there was new subchondral bone formation in both scaffolds. Short-term scaffolds had a statistically significant increase in defect filling and better qualitative histologic fill compared to control. Conclusions The PSPU short-term degradation scaffold may aid in cartilage repair by ultimately incorporating the scaffold into the microfracture procedure.

Download Full-text

Cartilage T2 Relaxation Times and Subchondral Trabecular Bone Parameters Predict Morphological Outcome After Matrix-Associated Autologous Chondrocyte Implantation With Autologous Bone Grafting

The American Journal of Sports Medicine ◽

10.1177/0363546520965987 ◽

2020 ◽

Vol 48 (14) ◽

pp. 3573-3585

Author(s):

Alexandra S. Gersing ◽

Christian Holwein ◽

Joachim Suchowierski ◽

Georg Feuerriegel ◽

Florian T. Gassert ◽

...

Keyword(s):

Mr Imaging ◽

Trabecular Bone ◽

Cartilage Repair ◽

Subchondral Bone ◽

Clinical Symptoms ◽

Repair Tissue ◽

Bone Parameters ◽

Bone Fraction ◽

Chondrocyte Implantation ◽

Trabecular Number

Background: Quantitative magnetic resonance (MR) imaging techniques are established for evaluation of cartilage composition and trabecular bone microstructure at the knee. It remains unclear whether quantitative MR parameters predict the midterm morphological outcome after matrix-associated chondrocyte implantation (MACI) with autologous bone grafting (ABG). Purpose: To assess longitudinal changes and associations of the biochemical composition of cartilage repair tissue, the subchondral bone architecture, and morphological knee joint abnormalities on 3-T MR imaging after MACI with ABG at the knee. Study Design: Case series; Level of evidence, 4. Methods: Knees of 18 patients (28.7 ± 8.4 years [mean ± SD]; 5 women) were examined preoperatively and 3, 6, 12, and 24 months after MACI and ABG using 3-T MR imaging. Cartilage composition was assessed using T2 relaxation time measurements. Subchondral bone microstructure was quantified using a 3-dimensional phase-cycled balanced steady-state free precision sequence. Trabecular bone parameters were calculated using a dual threshold algorithm (apparent bone fraction, apparent trabecular number, and apparent trabecular separation). Morphological abnormalities were assessed using the MOCART (magnetic resonace observation of cartilage repair tissue) score, the WORMS (Whole-Organ Magnetic Resonance Imaging Score), and the CROAKS (Cartilage Repair Osteoarthritis Knee Score). Clinical symptoms were assessed using the Tegner activity and Lysholm knee scores. Statistical analyses were performed by using multiple linear regression analysis. Results: Total WORMS ( P = .02) and MOCART ( P = .001) scores significantly improved over 24 months after MACI. Clinical symptoms were significantly associated with the presence of bone marrow edema pattern abnormalities 24 months after surgery ( P = .035). Overall there was a good to excellent radiological outcome found after 24 months (MOCART score, 88.8 ± 10.1). Cartilage repair T2 values significantly decreased between 12 and 24 months after MACI ( P = .009). Lower global T2 values after 3 months were significantly associated with better MOCART scores after 24 months ( P = .04). Moreover, trabecular bone parameters after 3 months were significantly associated with the total WORMS after 24 months (apparent bone fraction, P = .048; apparent trabecular number, P = .013; apparent trabecular separation, P = .013). Conclusion: After MACI with ABG, early postoperative quantitative assessment of biochemical composition of cartilage and microstructure of subchondral bone may predict the outcome after 24 months. The perioperative global joint cartilage matrix quality is essential for proper proliferation of the repair tissue, reflected by MOCART scores. The subchondral bone quality of the ABG site is essential for proper maturation of the cartilage repair tissue, reflected by cartilage T2 values.

Download Full-text

Isolation, Culture, and Characterization of Chicken Cartilage Stem/Progenitor Cells

BioMed Research International ◽

10.1155/2015/586290 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 1

Author(s):

Lu Li ◽

Yuehui Ma ◽

Xianglong Li ◽

Xiangchen Li ◽

Chunyu Bai ◽

...

Keyword(s):

Articular Cartilage ◽

Progenitor Cells ◽

Cartilage Repair ◽

Growth Curves ◽

Cell Types ◽

Biological Characteristics ◽

Surface Zone ◽

Multipotent Stem Cells

A chondrocyte progenitor population isolated from the surface zone of articular cartilage has become a promising cell source for cell-based cartilage repair. The cartilage-derived stem/progenitor cells are multipotent stem cells, which can differentiate into three cell types in vitro including adipocytes, osteoblasts, and chondrocytes. Much work has been done on cartilage stem/progenitor cells (CSPCs) from people, horses, and cattle, but the relatively little literature has been published about these cells in chickens. In our work, CSPCs were isolated from chicken embryos in incubated eggs for 20 days. In order to inquire into the biological characteristics of chicken CSPCs, immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry were adopted to detect the characteristic surface markers of CSPCs. Primary CSPCs were subcultured to passage 22 and, for purpose of knowing the change of cell numbers, we drew the growth curves. Isolated CSPCs were induced to adipocytes, osteoblasts, and chondrocytes. Our results suggest that we have identified and characterised a novel cartilage progenitor population resident in chicken articular cartilage and CSPCs isolated from chickens possess similar biological characteristics to those from other species, which will greatly benefit future cell-based cartilage repair therapies.

Download Full-text

Surface-Coated Polylactide Fiber Meshes as Tissue Engineering Matrices with Enhanced Cell Integration Properties

International Journal of Polymer Science ◽

10.1155/2014/439784 ◽

2014 ◽

Vol 2014 ◽

pp. 1-12 ◽

Cited By ~ 11

Author(s):

Matthias Schnabelrauch ◽

Ralf Wyrwa ◽

Henrike Rebl ◽

Claudia Bergemann ◽

Birgit Finke ◽

...

Keyword(s):

Bone Cells ◽

Polymer Network ◽

Fiber Surface ◽

Cell Types ◽

Structural Features ◽

Tissue Response ◽

Hydrophobic Nature ◽

Positively Charged

Poly(L-lactide-co-D/L-lactide)-based fiber meshes resembling structural features of the native extracellular matrix have been prepared by electrospinning. Subsequent coating of the electrospun fibers with an ultrathin plasma-polymerized allylamine (PPAAm) layer after appropriate preactivation with continuous O2/Ar plasma changed the hydrophobic nature of the polylactide surface into a hydrophilic polymer network and provided positively charged amino groups on the fiber surface able to interact with negatively charged pericellular matrix components. In vitro cell experiments using different human cell types (epithelial origin: gingiva and uroepithelium; bone cells: osteoblasts) revealed that the PPAAm-activated surfaces promoted the occupancy of the meshes by cells accompanied by improved initial cell spreading. This nanolayer is stable in its cell adhesive characteristics also afterγ-sterilization. An in vivo study in a rat intramuscular implantation model demonstrated that the local inflammatory tissue response did not differ between PPAAm-coated and untreated polylactide meshes.

Download Full-text

A tri-component knee plug for the 3rd generation of autologous chondrocyte implantation

Scientific Reports ◽

10.1038/s41598-020-73863-x ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Lobat Tayebi ◽

Zhanfeng Cui ◽

Hua Ye

Keyword(s):

Autologous Chondrocyte Implantation ◽

Sodium Hyaluronate ◽

Porous Membrane ◽

Knee Cartilage ◽

Permeable Membrane ◽

Chondrocyte Implantation ◽

3D Printed ◽

Surface Modified ◽

Autologous Chondrocyte

Abstract Here, we report a newly designed knee plug to be used in the 3rd generation of Autologous Chondrocyte Implantation (ACI) in order to heal the damaged knee cartilage. It is composed of three components: The first component (Bone Portion) is a 3D printed hard scaffold with large pores (~ 850 µm), made by hydroxyapatite and β-tricalcium phosphate to accommodate the bony parts underneath the knee cartilage. It is a cylinder with a diameter of 20 mm and height of 7.5 mm, with a slight dome shape on top. The plug also comprises a Cartilage Portion (component 2) which is a 3D printed gelatin/elastin/sodium-hyaluronate soft thick porous membrane with large pores to accommodate chondrocytes. Cartilage Portion is secured on top of the Bone Portion using mechanical interlocking by designing specific knobs in the 3D printed construct of the Cartilage Portion. The third component of the plug (Film) is a stitchable permeable membrane consisting of polycaprolactone (PCL) on top of the Cartilage Portion to facilitate sliding of the knee joint and to hold the entire plug in place while allowing nutrients delivery to the Cartilage Portion. The PCL Film is prepared using a combination of film casting and sacrificial material leaching with a pore size of 10 µm. It is surface modified to have specific affinity with the Cartilage Portion. The detailed design criteria and production process of this plug is presented in this report. Full in vitro analyses have been performed, which indicate the compatibility of the different components of the plug relative to their expected functions.

Download Full-text

In vitro proliferation and lifespan of bovine aorta endothelial cells: response to conditioned media

Journal of Cell Science ◽

10.1242/jcs.56.1.281 ◽

1982 ◽

Vol 56 (1) ◽

pp. 281-292

Author(s):

D.W. Lincoln ◽

R.G. Whitney ◽

J.R. Smith

Keyword(s):

Growth Rate ◽

Endothelial Cells ◽

Conditioned Medium ◽

Cell Types ◽

Conditioned Media ◽

In Vitro Proliferation ◽

Present Evidence ◽

Growth Promoting ◽

Population Doublings

The effect of conditioned medium on the growth rate of bovine aorta endothelial cells (CSC311) was examined. Media conditioned by several cell types (both immortal and normal) was found to increase the growth rate of CSC311 cells. The growth rate of CSC311 was increased even when the medium was conditioned by other CSC311 cells. The rate of growth of unstimulated CSC311 cells is 0.43 population doublings (p.d./day. Conditioned medium from several cell types gave a maximum stimulated growth rate of 1.3 p.d./day. In addition, we present evidence for an increase in the degree of stimulation by the growth-promoting activity found in conditioned medium with increasing in vitro age of CSC311 cells.

Download Full-text

Combined Optimized Effect of a Highly Self-Organized Nanosubstrate and an Electric Field on Osteoblast Bone Cells Activity

BioMed Research International ◽

10.1155/2019/7574635 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Diana V. Portan ◽

Despina D. Deligianni ◽

George C. Papanicolaou ◽

Vassilis Kostopoulos ◽

Georgios C. Psarras ◽

...

Keyword(s):

Electrical Stimulation ◽

Electric Field ◽

Bone Cells ◽

Electrical Field Stimulation ◽

Cell Types ◽

Specific Cell ◽

Specific Substrate ◽

Future Design ◽

Protein Levels

The effect of an electric field within specific intensity limits on the activity of human cells has been previously investigated. However, there are a considerable number of factors that influence the in vitro development of cell populations. In biocompatibility studies, the nature of the substrate and its topography are decisive in osteoblasts bone cells development. Further on, electrical field stimulation may activate biochemical paths that contribute to a faster, more effective self-adjustment and proliferation of specific cell types on various nanosubstrates. Within the present research, an electrical stimulation device has been manufactured and optimum values of parameters that led to enhanced osteoblasts activity, with respect to the alkaline phosphatase and total protein levels, have been found. Homogeneous electric field distribution induced by a highly organized titanium dioxide nanotubes substrate had an optimum effect on cell response. Specific substrate topography in combination with appropriate electrical stimulation enhanced osteoblasts bone cells capacity to self-adjust the levels of their specific biomarkers. The findings are of importance in the future design and development of new advanced orthopaedic materials for hard tissue replacement.

Download Full-text