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Comparative Study
. 2019 Jul;47(9):2188-2199.
doi: 10.1177/0363546519855645.

Dynamic Compressive Loading Improves Cartilage Repair in an In Vitro Model of Microfracture: Comparison of 2 Mechanical Loading Regimens on Simulated Microfracture Based on Fibrin Gel Scaffolds Encapsulating Connective Tissue Progenitor Cells

Tomoya Iseki  1   2 , Benjamin B Rothrauff  1 , Shinsuke Kihara  1 , Hiroshi Sasaki  1 , Shinichi Yoshiya  2 , Freddie H Fu  3 , Rocky S Tuan  1   4 , Riccardo Gottardi  1   5
Affiliations
Comparative Study

Dynamic Compressive Loading Improves Cartilage Repair in an In Vitro Model of Microfracture: Comparison of 2 Mechanical Loading Regimens on Simulated Microfracture Based on Fibrin Gel Scaffolds Encapsulating Connective Tissue Progenitor Cells

Tomoya Iseki et al. Am J Sports Med. 2019 Jul.

Abstract

Background: Microfracture of focal chondral defects often produces fibrocartilage, which inconsistently integrates with the surrounding native tissue and possesses inferior mechanical properties compared with hyaline cartilage. Mechanical loading modulates cartilage during development, but it remains unclear how loads produced in the course of postoperative rehabilitation affect the formation of the new fibrocartilaginous tissue.

Purpose: To assess the influence of different mechanical loading regimens, including dynamic compressive stress or rotational shear stress, on an in vitro model of microfracture repair based on fibrin gel scaffolds encapsulating connective tissue progenitor cells.

Study design: Controlled laboratory study.

Methods: Cylindrical cores were made in bovine hyaline cartilage explants and filled with either (1) cartilage plug returned to original location (positive control), (2) fibrin gel (negative control), or (3) fibrin gel with encapsulated connective tissue progenitor cells (microfracture mimic). Constructs were then subjected to 1 of 3 loading regimens: (1) no loading (ie, unloaded), (2) dynamic compressive loading, or (3) rotational shear loading. On days 0, 7, 14, and 21, the integration strength between the outer chondral ring and the central insert was measured with an electroforce mechanical tester. The central core component, mimicking microfracture neotissue, was also analyzed for gene expression by real-time reverse-transcription polymerase chain reaction, glycosaminoglycan, and double-stranded DNA contents, and tissue morphology was analyzed histologically.

Results: Integration strengths between the outer chondral ring and central neotissue of the cartilage plug and fibrin + cells groups significantly increased upon exposure to compressive loading compared with day 0 controls (P = .007). Compressive loading upregulated expression of chondrogenesis-associated genes (SRY-related HGMG box-containing gene 9 [SOX9], collagen type II α1 [COL2A1], and increased ratio of COL2A1 to collagen type I α1 [COL1A1], an indicator of more hyaline phenotype) in the neotissue of the fibrin + cells group compared with the unloaded group at day 21 (SOX9, P = .0032; COL2A1, P < .0001; COL2A1:COL1A1, P = .0308). Fibrin + cells constructs exposed to shear loading expressed higher levels of chondrogenic genes compared with the unloaded condition, but the levels were not as high as those for the compressive loading condition. Furthermore, catabolic markers (MMP3 and ADAMTS 5) were significantly upregulated by shear loading (P = .0234 and P < .0001, respectively) at day 21 compared with day 0.

Conclusion: Dynamic compressive loading enhanced neotissue chondrogenesis and maturation in a simulated in vitro model of microfracture, with generation of more hyaline-like cartilage and improved integration with the surrounding tissue.

Clinical relevance: Controlled loading after microfracture may be beneficial in promoting the formation of more hyaline-like cartilage repair tissue; however, the loading regimens applied in this in vitro model do not yet fully reproduce the complex loading patterns created during clinical rehabilitation. Further optimization of in vitro models of cartilage repair may ultimately inform rehabilitation protocols.

Keywords: articular cartilage; connective tissue progenitor cells; mechanical loading; microfracture; rehabilitation.

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Conflict of interest statement

One or more of the authors has declared the following potential conflict of interest or source of funding: R.S.T. received the MATE system free of charge as a beta tester from Apex Biomedical LLC (materials from a company that might benefit from this study). This research received funding from the Alliance for Regenerative Rehabilitation Research and Training (AR3T), which is supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), National Institute of Neurological Disorders and Stroke (NINDS), and National Institute of Biomedical Imaging and Bioengineering (NIBIB) of the National Institutes of Health (NIH) under award number P2CHD086843. This work is also supported, in part, by the US Department of Defense (W81XWH-14-2-0003 to R.S.T), by the EU Horizon 2020 - Research and Innovation Action SC1-PM 17 - 2017 +Project OACTIVE (under Grant Agreement No. 777159 to R.G.), and by Fondazione Ri.MED (grant to R.G.). AOSSM checks author disclosures against the Open Payments Database (OPD). AOSSM has not conducted an independent investigation on the OPD and disclaims any liability or responsibility relating thereto.

Figures

Figure 1.
Figure 1.
Schematic of experimental design. The in vitro model of microfracture consisted of articular cartilage plugs harvested from bovine knees, which were then centrally cored out to form a cylindrical defect space, followed by implantation of 1 of the following 3 experimental constructs: (1) positive control (inner plug returned to the defect), (2) negative control (fibrin only), and (3) microfracture mimic (fibrin + cells). These composite constructs were then exposed to 1 of 3 mechanical loading regimens: (1) unloaded, (2) compressive loading (MechanoActive Transduction and Evaluation bioreactor; MATE), and (3) shear loading (rotatory cell culture system; RCCS). At the end of the indicated experimental periods, some samples (n = 2 per group, per time point) were processed for histological analysis, and the remaining samples (n = 4) were evaluated by push-out test. After testing, half of the samples were processed for gene expression profiling by quantitative reverse-transcription polymerase chain reaction (n = 2) and for biochemical analysis (n = 2). TGF-β3, transforming growth factor β3.
Figure 2.
Figure 2.
Mechanical loading of cartilage microfracture models in culture. (A) Schematic image of MechanoActive Transduction and Evaluation (MATE) bioreactor. The electromagnetic coil motor (VCM) raises the plunger and culture dish, thereby compressing specimens onto impermeable posts. Adapted with permission from Lujan TJ, Wirtz KM, Bahney CS, Madey SM, Johnstone B, Bottlang M. A novel bioreactor for the dynamic stimulation and mechanical evaluation of multiple tissue-engineered constructs. Tissue Eng Part C Methods. 2011;17(3):367-374. (B) The compact-sized MATE readily fits into standard incubators. (C) Rotatory cell culture system in the incubator.
Figure 3.
Figure 3.
Measurement of integration strength between central core and outer hyaline cartilage ring. (A) Depiction of cartilage microfracture model and photograph of push-out test setup. (B) Cross-sectional schematics of the push-out test setup. Integration strength was calculated as the ratio of the maximum force registered over the contact surface between the outer ring and inner core.
Figure 4.
Figure 4.
Integration between outer cartilage ring and central insert. (A-C) Integration strength between outer hyaline chondral ring and central insert of (A) cartilage plug, (B) fibrin only, and (C) fibrin + cells, compared with day 0 (control), P < .05. (D-F) Safranin O/Fast Green histological staining of the interface between outer hyaline chondral ring and central insert: (D) cartilage plug, (E) fibrin only, and (F) fibrin + cells at day 14 in the compressive loading condition. Arrowheads indicate pericellular deposition of proteoglycan. Bar, 100 µm; n = 12, combined from 3 independent trials.
Figure 5.
Figure 5.
Safranin-O/Fast Green staining of fibrin with cells. (A) Day 0 controls and days 7 and 21 by loading condition; scale bar = 200 µm. Glycosaminoglycan (GAG) stained red, and fibrin stained blue/green, showing higher GAG content in unloaded and compressive loading conditions at day 21. (B) Normalized glycosaminoglycan content (GAG/DNA) as a function of culture time by loading condition. *Significantly higher than day 0 controls (P < .05).
Figure 6.
Figure 6.
Gene expression of fibrin + cells group cultured under 3 mechanical loading conditions. Gene expression was analyzed by quantitative reverse-transcription polymerase chain reaction for (A) SOX9, (B) COL2A1, (C) ACAN, (D) COL2:COL1 (ratio of collagen type II to collagen type I), (E) MMP3, and (F) ADAMTS5. All expression levels are expressed relative to day 0 controls. *Comparison with day 0 controls (P < .05). **Comparison with other time points within the same activation group (P < .05). #Comparison between activation groups at the same time point (P < .05). ACAN, aggrecan; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; COL1A1, collagen type I α1; MMP, matrix metalloproteinase; SOX9, SRY-related HGMG box containing gene 9.
Figure 7.
Figure 7.
Elastic moduli of composite constructs at days 0 and 21. (A) cartilage plug, (B) fibrin only, and (C) fibrin + cells. *P < .05.
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