Immunohistochemical Analysis of Knee Chondral Defect Repair after Autologous Particulated Cartilage and Platelet-Rich Plasma Treatment in Sheep

Abstract

This study performs an analysis that will enable the evaluation of the quality, durability, and structure of repaired cartilaginous extracellular matrix tissue using an autologous-based particulated autograft cartilage and platelet-rich plasma treatment (PACI + PRP). A single-blind controlled experiment was conducted on 28 sheep to evaluate the efficacy of the PACI + PRP treatment for cartilage defects. Full-thickness 8 mm diameter defects were created in the weight-bearing area of both knees. The right knees received PACI + PRP. The left knees were treated with Ringer's lactate solution (RLS) or hyaluronic acid (HA) injections. Sheep were euthanized at 9- or 18-months post-surgery. An extensive immunohistochemical analysis was performed to assess collagen types (I, II, III, V, VI, IX, X, XI) and aggrecan positivity. A semiquantitative scoring system provided a detailed evaluation of immunostaining. Collagens and aggrecan scores in the PACI + PRP groups were similar to healthy cartilage. Significant differences were found in collagens associated with matrix maturity (II and V), degradation (IX), structure and mechanics (VI), and hypertrophy (X) between healthy cartilage and RLS- or HA-repaired cartilage. The PACI + PRP treatment advanced the repair cartilage process in chondral defects with mature hyaline cartilage and enhanced the structural and mechanical qualities with better consistent cartilage, less susceptible to degradation and without hypertrophic formation over time.

Keywords: chondral defect; immunohistochemical; knee; particulated cartilage; platelet-rich plasma.

Figure 1

Immunostaining scores of collagen types and aggrecan studied for RLS, PACI + PRP(RLS), HA, and PACI + PRP(HA) at 9 months. (A) Type I collagen; (B) type II collagen; (C) type III collagen; (D) type V collagen; (E) type VI collagen; (F) type IX collagen; (G) type X collagen; (H) type XI collagen; (I) aggrecan. RLS: left knee treated with Ringer’s lactate solution; PACI + PRP(RLS): right knee treated with particulated autograft cartilage implantation and platelet-rich plasma from the group treated with RLS in the left knee; HA: left knee treated with hyaluronic acid; PACI + PRP(HA): right knee treated with particulated autograft cartilage implantation and platelet-rich plasma from the group treated with HA in the left knee. * Significant difference between groups, p < 0.05.

Figure 2

Immunostaining scores of collagen types and aggrecan studied for RLS, PACI + PRP(RLS), HA, and PACI + PRP(HA) at 18 months. (A) type I collagen; (B) type II collagen; (C) type III collagen; (D) type V collagen; (E) type VI collagen; (F) type IX collagen; (G) type X collagen; (H) type XI collagen; (I) aggrecan. RLS: left knee treated with Ringer’s lactate solution; PACI + PRP(RLS): right knee treated with particulated autograft cartilage implantation and platelet-rich plasma from the group treated with RLS in the left knee; HA: left knee treated with hyaluronic acid; PACI + PRP(HA): right knee treated with particulated autograft cartilage implantation and platelet-rich plasma from the group treated with HA in the left knee. * Significant difference between groups, p < 0.05.

Figure 3

Immunohistochemical evaluation of healthy cartilage and repaired tissue at 9 months after the administered treatments. (A) Evaluation of type II collagen in HA group at 9 months; (B) example of healthy cartilage surrounding the chondral defect immunostained with type II collagen at 9 months; (C) evaluation of type II collagen in the PACI + PRP group at 9 months; (D) evaluation of type V collagen in the RLS group at 9 months; (E) example of healthy cartilage surrounding the chondral defect immunostained with type V collagen at 9 months; (F) evaluation of type V collagen in the PACI + PRP group at 9 months. RLS: left knee treated with Ringer’s lactate solution; HA: left knee treated with hyaluronic acid; PACI + PRP: right knee treated with particulated autograft cartilage implantation and platelet-rich plasma.

Figure 4

Immunohistochemical evaluation of healthy cartilage and repaired tissue at 18 months after the treatments administered. (A) Evaluation of type IX collagen in the HA group at 18 months; (B) example of healthy cartilage surrounding the chondral defect immunostained with type IX collagen at 18 months; (C) evaluation of type IX collagen in the PACI + PRP group at 18 months; (D) evaluation of type X collagen in the RLS group at 18 months; (E) example of healthy cartilage immunostained surrounding the chondral defect with type X collagen at 18 months; (F) evaluation of type X collagen in the PACI + PRP group at 18 months. RLS: left knee treated with Ringer’s lactate solution; HA: left knee treated with hyaluronic acid; PACI + PRP: right knee treated with particulated autograft cartilage implantation and platelet-rich plasma.

Figure 5

Immunohistochemical evaluation of healthy cartilage and the repaired tissue at 9 and 18 months after the treatments administered. (A) Evaluation of type VI collagen in the RLS group at 9 months; (B) example of healthy cartilage surrounding the chondral defect immunostained with type VI collagen at 9 months; (C) evaluation of type VI collagen in the PACI + PRP group at 9 months; (D) evaluation of type VI collagen in the RLS group at 18 months; (E) example of healthy cartilage surrounding the chondral defect immunostained with type VI collagen at 18 months; (F) evaluation of type VI collagen in the PACI + PRP group at 18 months; (G) evaluation of type VI collagen in the HA group at 9 months; (H) example of healthy cartilage surrounding the chondral defect immunostained with type VI collagen at 9 months; (I) evaluation of type VI collagen in the PACI + PRP group at 9 months; (J) evaluation of type VI collagen in the HA group at 18 months; (K) example of healthy cartilage surrounding the chondral defect immunostained with type VI collagen at 18 months; (L) evaluation of type VI collagen in the PACI + PRP group at 18 months. RLS: left knee treated with Ringer’s lactate solution; HA: left knee treated with hyaluronic acid; PACI + PRP: right knee treated with particulated autograft cartilage implantation and platelet-rich plasma.