Original Article
Supplementation With Platelet-Rich Plasma Improves the In Vitro Formation of Tissue-Engineered Cartilage With Enhanced Mechanical Properties

https://doi.org/10.1016/j.arthro.2013.07.259Get rights and content

Purpose

This study aimed to determine the effects of platelet-rich plasma (PRP) on the histologic, biochemical, and biomechanical properties of tissue-engineered cartilage.

Methods

Chondrocytes isolated from bovine metacarpal-phalangeal articular cartilage were seeded on top of a porous ceramic substrate (calcium polyphosphate [CPP]). Cultures were supplemented with fetal bovine serum (FBS), PRP, or platelet-poor plasma (PPP) at 5%. On day 5, the concentration was increased to 20%. PRP and PPP were obtained through centrifugation of whole blood withdrawn from a mature cow. After 2 weeks, samples (n = 8) were analyzed histologically, biochemically, and biomechanically. Data were analyzed using the Wilcoxon test (significance, P < .05).

Results

Chondrocytes cultured in 20% PRP formed thicker cartilage tissue (1.6 ± 0.2 mm) than did cells grown in 20% FBS (0.7 ± 0.008 mm; P = .002) and 20% PPP (0.8 ± 0.2 mm; P = .03). Cartilage tissue generated in the presence of 20% PRP had a greater equilibrium modulus of 38.1 ± 3.6 kPa versus 15.6 ± 1.5 kPa (P = .0002) for 20% PPP and 20.4 ± 3.5 kPa (P = .007) for 20% FBS. Glycosaminoglycan (GAG) content was increased in tissues formed in 20% PRP (176 ± 18.8 μg GAG/mg) compared with those grown in 20% FBS (112 ± 10.6 μg GAG/mg; P = .01) or 20% PPP (131.5 ± 14.8 μg GAG/mg; P = .11). Hydroxyproline content was similar whether the media was supplemented with 20% PRP (8.7 ± 0.9 μg/mg), 20% FBS (7.6 ± 0.9 μg/mg; P = .37), or 20% PPP (6.4 ± 1 μg/mg; P = .28). DNA content was similar in all tissues whether formed in 20% PRP (11.9 ± 3.5 μg/mg), 20% FBS (9.3 ± 2.5 μg/mg; P = .99), or 20% PPP (7.2 ± 1.3 μg/mg; P = .78). Immunostained samples showed prevalence of type II collagen in tissues formed in the presence of 20% PRP.

Conclusions

The presence of PRP in the culture media enhances the in vitro formation of cartilage, with increased GAG content and greater compressive mechanical properties, while maintaining characteristics of hyaline phenotype.

Clinical Relevance

Understanding the in vitro effects of PRP on tissue-engineered cartilage may lead to the creation of engineered cartilage tissue with enhanced properties suitable for cartilage repair.

Section snippets

PRP and Platelet-Poor Plasma Preparation

Bovine blood was collected from the jugular vein of a mature cow in 6 60-mL syringes with citrate dextrose solution as anticoagulant (1.3 mL/10 mL of blood). Whole blood was centrifuged at 1,000 rpm for 10 minutes using a Sorvall RT7 centrifuge (Mandel Scientific, Guelph, ONT, Canada), leading to separation of plasma and platelets from red blood cells. Plasma was then collected and further centrifuged at 3,000 rpm for 5 minutes, producing a concentrate of platelets or PRP. The supernatant of

Platelet Count

PRP had greater platelet numbers than did the PPP fraction. The concentration of platelets in PRP was 1.22 × 106/mL, whereas in PPP it was 0.03 × 106/mL.

Mechanical Properties

The samples cultured with 20% PRP exhibited the highest Young's modulus, with an equilibrium modulus equal to 38.1 ± 3.6 kPa. Constructs cultured with 20% PPP and 20% FBS had inferior equilibrium moduli (15.6 ± 1.5 kPa; P = .0002 and 20.4 ± 3.5 kPa; P = .007, respectively) (Table 1).

Macroscopic Evaluation and Histologic Examination

Gross observation of the 3 constructs revealed that samples

Discussion

Tissue-engineered cartilage implants are a novel strategy for the treatment of cartilage injuries. This study shows that a tissue-engineered biphasic implant cultured in media supplemented with 20% PRP had significantly thicker cartilage tissue, with increased ECM content and improved mechanical properties compared with implants cultured with 20% FBS or 20% PPP. The increase in matrix predominantly resulted from increased proteoglycan content.

The results of our study are consistent with other

Conclusions

The presence of PRP in the culture media enhances the in vitro formation of cartilage with increased GAG content and greater compressive mechanical properties while maintaining characteristics of the hyaline phenotype.

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    The authors report the following potential conflict of interest or source of funding in relation to this article: M.H. serves on the board of, and has received an operating grant from, Canadian Arthritis Network, and has received payment for educational seminars/laboratories from International Cartilage Repair Society; J.S.T. has received payment for consultancy from Zimmer and Smith & Nephew Canada.

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