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Kartogenin Combined Platelet-Rich Plasma (PRP) Promoted Tendon-Bone Healing for Anterior Cruciate Ligament (ACL) Reconstruction by Suppressing Inflammatory Response Via targeting AKT/PI3K/NF-κB

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Abstract

Anterior cruciate ligament (ACL) rupture is the most common sports injuries and PRP has the potential to be a kartogenin (KGN) carrier to promote collagen fibril organization and cartilage regenerative in the tendon-bone interface. This paper aimed to investigate co-injection of KGN-PRP into the bone tunnels of ACL reconstructions which could enhance tendon-bone healing graft osteointegration effectively. HPLC was used to measured release rate of KGN from KGN-PRP gel. Then, an ACL injury reconstruction model in rabbits was established and the rabbits received saline, PRP, and KGN-PRP injection onto the tendon-bone interface after reconstruction. The tissue was harvested from the tendon-bone interface at 4 weeks and 8 weeks post-surgery, and the sections were stained with Safranin O/fast green to detected tendon-bone healing. Immunochemistry staining was used to analyze VEGF, collagen I, and HIF-1α expression, and ELISA assay was used for detecting IL-6, TNF-α, and COX-2 concentrations. The expression levels of AKT/PI3K/NF-κB-related protein and mRNA were presented by Western blot and qPCR. The release rate of KGN was high within 4 h of KGN-PRP gel and followed by a slow release until 7 days. The Safranin O/fast green staining results indicated that tendon-bone interface in sham and mock group existed gap and tissue disorganization. The KGN + PRP group showed the positive color of the healing interface was more obvious and cartilage tissue began to be generated in large amounts at this interface. The maximum tensile force of KGN-PRP injection tendon-bone healing site was significantly higher than that of PRP group, and KGN-PRP effectively promoted fibro chondrogenesis and tendon-bone healing. The expression of collagen I, VEGF, and HIF-1α in regenerated tissues at the healing interface was significantly increased by KGN-PRP treatment compared with the mock and sham groups. The expressions of IL-6, TNF-α, and COX-2 after KGN-PRP treatment were significantly decreased in tendon-bone interface compared to the mock group. WB and qPCR results showed KGN-PRP treatment effectively inhibits AKT/PI3K/NF-κB activation of inflammatory pathways, thereby reducing the level of inflammation to promote wound healing. PRP is an effective carrier for KGN with the sustained release of KGN. After ACL reconstruction, injection of KGN-PRP gel significantly reduced the inflammatory response and inhibited AKT/PI3K/NF-κB activation in cartilage tissue, which promoted tendon-bone healing.

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Abbreviations

PRP:

Platelet-rich plasma

ACL:

Anterior cruciate ligament

PI3K:

Phosphatidylinositol-3 kinase

AKT/PKB:

Protein kinase B

NF-κB:

Nuclear factor-kappa B

KGN:

Kartogenin

HPLC:

High-performance liquid chromatography

VEGF:

Vascular endothelial growth factor

HIF1α:

Hypoxia-inducible factor-1α

ELISA:

Enzyme-linked immunosorbent assay

IL-6:

Interleukin 6

TNF-α:

Tumor necrosis factor-alpha

COX-2:

Cyclooxygenase-2

WB:

Western blotting

qPCR:

Quantitative polymerase chain reaction

PBS:

Phosphate-buffered saline

EDTA:

Ethylenediaminetetraacetic acid

BCA:

Bicinchoninic acid

PVDF:

Polyvinylidene fluoride

hASCs:

Human adipose stromal cells

rhBMP-2:

Recombinant human bone morphogenetic protein-2

IL-1β:

Interleukin-1 beta

ROS:

Reactive oxygen species

OA:

Osteoarthritis

SOX9:

SRY-box transcription factor 9

Coll2:

Type II collagen

ACAN:

Aggrecan

MMP13:

Matrix metallopeptidase-13

ERK:

Extracellular signal–regulated kinase

ANGPTL4:

Angiopoetin-like 4

TGF-β:

Transforming growth factor beta

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Authors and Affiliations

  1. Department of Orthopedics Surgery, The Third Hospital of Hebei Medical University, Shijiazhuang, 050051, Hebei, People’s Republic of China

    Lei Zhang, Qian Zhang, Lukuan Cui, Lijie Wu & Shijun Gao

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  1. Lei Zhang
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  2. Qian Zhang
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  3. Lukuan Cui
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  4. Lijie Wu
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All authors contributed equally.

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Correspondence to Shijun Gao.

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The authors’ institution provided ethical approval for the conduct of this study 2022–091.

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Zhang, L., Zhang, Q., Cui, L. et al. Kartogenin Combined Platelet-Rich Plasma (PRP) Promoted Tendon-Bone Healing for Anterior Cruciate Ligament (ACL) Reconstruction by Suppressing Inflammatory Response Via targeting AKT/PI3K/NF-κB. Appl Biochem Biotechnol 195, 1284–1296 (2023). https://doi.org/10.1007/s12010-022-04178-y

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