Table 2.

Impact of sex hormones on cartilage regeneration

Hormones	Study design	Treatment	Analysis	Results	Implications	References
T/DHT	In vitro; murine ESCs	Cultured with T, DHT, or NT at each of the following concentrations: 10–6 M, 10–8 M, and 10–10 M in ethanol	IHC for androgen receptor, qPCR, cell colorimetric assays, Western blot	T had no significant effects on proliferation, while DHT marginally inhibited proliferation of ESCs; NT significantly increased ESC proliferation	ESC proliferation from blastocyst is independent of androgen effects	[207]
T	In vitro; human IVD cells and BMSCs	Differentiation in the presence or absence of T (10 ng/mL)	PCR, immunoblotting, IHC	Male IVD cells exposed to T exhibited increased aggrecan and types I and II collagen expression compared to control; T had no effect on female IVD cell chondrogenesis or BMSC chondrocytes from either sex	T increases ECM production in male IVD cells during differentiation	[92]
	In vivo; male rabbit growth plate chondrocytes	ORX versus non-ORX male rabbits	Soft radiography, IHC, quantification of caspase-3 and PCNA expression	At weeks 8–15 for ORX group, proliferation was decreased in quantity and displayed a narrowed proliferating zone compared to control	T accelerates growth and maturation of the epiphyseal plate and stimulates proliferation in males	[206]
	In vitro; rat costochondral growth zone and resting zone chondrocytes	Incubation with various doses of T: 10,100, and 1000 ng/mL	Thymidine incorporation, cell quantity, ALP activity, and collagen production	T inhibited cell number and dose-dependent decrease in thymidine incorporation of male growth zone and resting zone chondrocytes; cell quantity and thymidine incorporation unaffected by T in female rat cells; only significant change in ALP activity was an increase in ALP activity in T-treated growth zone male chondrocytes; T stimulated production of collagen in male growth zone and resting zone chondrocytes but T did not affect female cell collagen production	T primarily influences male chondrocytes and induces osteoblastic and chondrogenic differentiation	[197]
	In vivo; human patients with severe knee OA	Unilateral TKR	Serum T levels, knee radiograph, and WOMAC pain/function analysis 6–8 weeks after surgery	On the operative knee, higher T levels were associated with less pain in both sexes; in the non-operative knee, higher T in women was associated with less disability	T is positively correlated with less pain after TKR in both sexes and less disability in women without TKR	[196]
E2	In vitro; human ASCs	Cultured with E2 (concentration of 10–8 M) or without E2	RT-PCR	E2 exposure led to inhibition of COL2A1 expression and reduction of ACAN expression	E2 may have a negative impact on human ASC chondrogenesis	[216]
	In vitro; human BMSCs	Cultured with E2 (concentrations ranging from 10–11 M to 10–8 M) with or without ER inhibitor	Histology, IHC, quantification of type II collagen, sulfated GAGs, type X collagen, and MMP13	E2 treatment resulted in a reduction of type II collagen and enhancement of type X collagen and MMP13 expression	E2 suppresses chondrogenesis of human BMSCs	[215]
	In vitro; murine ESCs	Incubated in E2 (0–10−6 M for 8 h) or at 10–9 M for times ranging from 0 to 12 h	ALP staining, immunofluorescence, thymidine incorporation, BrdU incorporation, RT-PCR, Western blot	E2 treatment significantly increased thymidine incorporation, BrdU incorporation, and cell number	E2 stimulates proliferation of mouse ESCs	[208]
	In vitro; mini-pig BMSCs	Cultured in E2 at 0 M, 10–6 M, 10–8 M, 10–10 M, 10–12 M, or 10–14 M	Surface marker analysis, ALP, MTT proliferation assay, β-galactosidase staining, TUNEL staining, differentiation/cytochemical staining, RT-PCR	E2 increased proliferation in female BMSCs, with a dose of 10–12 M optimizing proliferation. E2 also increased proliferation in male cells to a lesser degree that is still significant; E2 at 10–12 M inhibited cellular senescence in BMSCs from both sexes; E2 did not significantly influence chondrogenic differentiation; E2 increased adipogenic differentiation ability in male BMSCs and osteogenic ability in female BMSCs	E2 improves cellular senescence and proliferation in both sexes	[210]
	In vitro; human articular chondrocytes	E2 treatment ranging from 10–11 to 10–7 M	Thymidine incorporation, ALP, RT-PCR for ER expression	E2-treated cells from female donors exhibited an increase in thymidine incorporation and ALP activity in a dose-dependent manner; no changes in thymidine incorporation or ALP activity seen in male cells with E2 treatment; no sex differences in ER expression when normalized to GAPDH	E2 stimulates proliferation and promotes osteogenic differentiation of female chondrocytes	[190]
	In vitro; female menopausal versus non-menopausal monkey articular chondrocytes	Transfection with a reporter construct containing ER element/luciferase	RT-PCR, immunoblotting, IHC	Articular cartilage contained functional ERs from both menopausal and non-menopausal donors	E2 receptors are functional after menopause, indicating ERT is functional after menopause	[189]
	In vivo; female monkey model	OVX female monkeys with and without ERT	Ligand blotting and immunoblotting to analyze production of IGFBPs; RT-PCR analysis for PG synthesis	IGFBP2 and PG expression was significantly increased in monkeys with ERT	ERT in menopausal females is chondroprotective	[189]

ASC adipose-derived stem cell, ALP alkaline phosphatase, BMSC bone-marrow-derived stem cells, BrDU bromodeoxyuridine, DHT dihydrotestosterone, E2 17-ß estradiol, ESC embryonic stem cell, ER estrogen receptor, ECM extracellular matrix, GAGs glycosaminoglycans, GAPDH glyceraldehyde 3-phosphate dehydrogenase, IGFBP insulin-like growth factor binding protein, IHC immunohistochemistry, IVD intervertebral disc, MMP13 matrix metalloproteinase 13, MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, NT antiandrogen nilutamide, OA osteoarthritis, ORX orchiectomy, OVX ovariectomy, PG progesterone, PCNA proliferating cell nuclear antigen, qPCR quantitative real-time polymerase chain reaction, RT-PCR reverse transcription polymerase chain reaction, TUNEL terminal deoxynucleotidyl transferase dUTP nick end labeling, T testosterone, TKR total knee replacement, WOMAC Western Ontario and McMaster Universities Osteoarthritis Index