(b).

Time of analysis	Activator	Results
72 h	Thrombin+CaCl2	Cell proliferation↑, PG and col synthesis↑, PG accumulation↑
7, 9 days	Thrombin	NP cell proliferation and aggregation↑; optimum at 1 ng TGF-β1 concentration in PRP. Tissue construct ↑ COL II, AGN, SOX-9 mRNA ↑, GAG accumulation ↑phosphorylation of Smad2/3 ↑, apoptosis ↓
4 weeks	Thrombin	NP regeneration ↑mRNA involved in chondrogenesis and matrix accumulation↑
7 days, 4 weeks	Acetic acid	Cell proliferation↑ chondrogenic differentiation↓
48 h	CaCl2	IL-1β and TNF-α led matrix synthesis gene expression↓; PRP degradation expression of COX-2 and MMP-3↓
7 days, 4 weeks	None	The expression of chondrogenic markers↑, inflammatory mediators ,matrix degrading enzymes in ihNPc↓
2, 4 days	Sonication	50% PL-DNA and GAG ↑. Matrix synthesis↑ of defect AF after PRP injection
1, 2, 3, 4, 5, 6, and 7 days	10% CaCl2 100 U thrombin	mRNA of COL II, AGN, and SOX-9↑; protein of COL X level TGF-b1/Smad2/3 ↑ COLII and AGN↑ by 2.5% PRP
24 h	1N HCL	COL II and AGN mRNA ↑,MMP1 mRNA, protein ↓
14 days	None	P-PRP: AGN, COL II↑, IL-1β, TNF-α, IL-6, IL-8, MMP-1, MMP-13 mRNA, IL-1β, TNF-α production ↓
7 days	10% CaCl2	P-PRP: AGN, COL II↑ L-PRP: IL-1β, TNF-α MMP1, MMP-13 mRNA ↑ NF-κB/p65 protein↑
0, 7, 14, 21 days	Freezing and thawing	Stimulated migration and cell viability in early COL II mRNA ↑, COL1 and 3 mRNA ↓

Abbreviations: ACT: activation; NP: nucleus pulposus; AF: annulus fibrosus; AGN: aggrecan; COL II: type II collagen; COX-2: cyclooxygenase-2; GAG: glycosaminoglycan; IVD: intervertebral disc; MMP: metalloproteinase; MSC: mesenchymal stem cell; Sox-9:SRY-related high mobility group box gene-9; PG: proteoglycan; PRP: platelet-rich plasma; P-PRP: leukocyte-poor PRP; R-PRP: leukocyte-rich PRP; TNF: tumor necrosis factor.