Sustained low-dose dexamethasone delivery via a PLGA microsphere-embedded agarose implant for enhanced osteochondral repair

Abstract

Articular cartilage defects are a common source of joint pain and dysfunction. We hypothesized that sustained low-dose dexamethasone (DEX) delivery via an acellular osteochondral implant would have a dual pro-anabolic and anti-catabolic effect, both supporting the functional integrity of adjacent graft and host tissue while also attenuating inflammation caused by iatrogenic injury. An acellular agarose hydrogel carrier with embedded DEX-loaded poly(lactic-co-glycolic) acid (PLGA) microspheres (DLMS) was developed to provide sustained release for at least 99 days. The DLMS implant was first evaluated in an in vitro pro-inflammatory model of cartilage degradation. The implant was chondroprotective, as indicated by maintenance of Young's modulus (E_Y) (p = 0.92) and GAG content (p = 1.0) in the presence of interleukin-1β insult. In a subsequent preliminary in vivo experiment, an osteochondral autograft transfer was performed using a pre-clinical canine model. DLMS implants were press-fit into the autograft donor site and compared to intra-articular DEX injection (INJ) or no DEX (CTL). Functional scores for DLMS animals returned to baseline (p = 0.39), whereas CTL and INJ remained significantly worse at 6 months (p < 0.05). DLMS knees were significantly more likely to have improved OARSI scores for proteoglycan, chondrocyte, and collagen pathology (p < 0.05). However, no significant improvements in synovial fluid cytokine content were observed. In conclusion, utilizing a targeted DLMS implant, we observed in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes. These improved outcomes were correlated with superior histological scores but not necessarily a dampened inflammatory response, suggesting a primarily pro-anabolic effect. STATEMENT OF SIGNIFICANCE: Articular cartilage defects are a common source of joint pain and dysfunction. Effective treatment of these injuries may prevent the progression of osteoarthritis and reduce the need for total joint replacement. Dexamethasone, a potent glucocorticoid with concomitant anti-catabolic and pro-anabolic effects on cartilage, may serve as an adjuvant for a variety of repair strategies. Utilizing a dexamethasone-loaded osteochondral implant with controlled release characteristics, we demonstrated in vitro chondroprotection in the presence of IL-1-induced degradation and improved in vivo functional outcomes following osteochondral repair. These improved outcomes were correlated with superior histological cartilage scores and minimal-to-no comorbidity, which is a risk with high dose dexamethasone injections. Using this model of cartilage restoration, we have for the first time shown the application of targeted, low-dose dexamethasone for improved healing in a preclinical model of focal defect repair.

Keywords: Dexamethasone; Microspheres; Osteochondral repair; Preclinical models; Targeted drug delivery.

Figure 1.

Study 1 Schematic. (A) Following a 42-day maturation period, engineered cartilage constructs (~200 kPa) were cored and implanted with DLMS or ULMS carriers for 3 days (Study 1a). (B) Mature engineered cartilage constructs were divided into six experimental groups for a 14-day interleukin-1β (IL) stimulation period where they were co-cultured with DLMS or ULMS carriers.

Figure 2.

(A) Schematic of Study 2 showing autograft donor site (i.), repair site (ii.), DLMS implant (iii.), and dexamethasone-PLGA microsphere release (iv.); (B) cartilage autograft (ii.) and DLMS implant (iii.); (C) autograft donor (i.) and repair (ii.) sites; (D) DLMS implant (iii.) and repair (ii.) sites.

Figure 3.

Representative immunohistochemical stain for p57^Kip2 expression (green) counterstained with DAPI. Inner core containing either (A) DLMS or (B) ULMS marked with * and boundary outlined with dotted line; (C) Relative pixel intensity of immunohistochemical stain expression as a function of distance away from the microsphere embedded core (n=1).

Figure 4.

(A) Young’s modulus (EY); (B) Dynamic modulus (G); (C) GAG/ww (%); (D) COL/ww (%); *p<0.05. (bars show mean and 95% CI).

Figure 5.

Representative safranin-o (saf-o) histology of the cartilage construct cross-section for (A) Day 42, (B) CTL ULMS, (C) CTL DLMS, (D) IL ULMS, (E) IL DLMS, and (F) corresponding relative staining intensity.

Figure 6.

(A-C) Gross imaging, (D-F) H&E, (G-I) toluidine blue, and (J-L) picrosirius red staining and (M-O) picrosirius red with polarized light for selected graft recipient sites; OATS-CTL (A, D, G, J, M; 92-FCL), OATS DLMS (B, E, H, K, N; 88-FCL), OATS-INJ (C, F, I, L, O; 112-FCL)

Figure 7.

Synovial Fluid Composition; *p<0.05 compared to Day 0 (same group), †p<0.05 compared to OATS CTL (same time point), ‡p<0.05 compared to OATS-INJ (same time point). (bars show median and 95% CI).

Figure 8.

(A) μCT revealed significant bone remodeling in the DLMS osteochondral implant; 30 (B) Reconstruction of DLMS bone base showing resorbed bone (pink), new bone (green), and unchanged bone (blue); Bone volume density (BV/TV), plate density (pBV/TV), rod density (rBV/TV), plate-rod ratio (PR ratio), number of plate trabeculi (pTb.N), number of rod trabeculi (rTb.N), plate trabecular thickness (pTb.Th), and rod trabecular thickness (rTb.Th); *p<0.05 vs. baseline.

Figure 9.

(A-C) Gross imaging, (D-F) H&E, (G-I) toluidine blue, and (J-L) picrosirius red staining and (M-O) picrosirius red with polarized light for selected graft donor sites; OATS-CTL (A, D, G, J, M; 92-FCL), OATS DLMS (B, E, H, K, N; 88-FCL), OATS-INJ (C, F, I, L, O; 112-FCL).