Acupotomy ameliorates subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis by regulating bone morphogenetic protein 2-Smad1 pathway

doi:10.19852/j.cnki.jtcm.20230404.001

Abstract

Abstract:

OBJECTIVE: To investigate the effects of acupotomy on the subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis (KOA).

METHODS: The rabbits were divided into blank control, model, acupotomy and electroacupuncture (EA) groups, with 12 rabbits in each. Modified Videman’s method was used to prepare KOA model. The acupotomy and EA group were given indicated intervention for 3 weeks. The behavior of rabbits in each group was recorded. Subsequently, cartilage-subchondral bone units were obtained and morphological changes were observed by optical microscope and micro computed tomography. Compression test was used to detect the mechanical properties of subchondral bone, Western blot and real-time polymerase chain reaction (RT-PCR) were applied to detect the expression of bone morphogenetic protein 2-Smad1 (BMP2-Smad1) pathway in subchondral bone.

RESULTS: Compared with the control group, rabbits in the KOA group showed lameness, knee pain, and cartilage degradation; the subchondral bone showed active resorption, the mechanical properties decreased significantly and the BMP2-Smad1 pathway downregulated significantly. Both acupotomy and EA intervention could increase the thickness of trabecular bone (Tb. Th), the bone volume fraction (BV/TV) and the thickness of subchondral bone plate, reduce the separation of trabecular bone (Tb. Sp), improve the maximum load and elastic modulus of subchondral bone, and effectively delay cartilage degeneration in KOA rabbits. This process may be achieved through upregulation the related proteins of BMP2-Smad1 pathway. The maximum load and elastic modulus of subchondral bone in the acupotomy group were slightly better than those in the EA group.

CONCLUSIONS: Acupotomy could effectively protect cartilage by inhibiting abnormal bone resorption and improving mechanical properties of subchondral bone thorough the related proteins of BMP2-Smad1 pathway in KOA rabbits.

Key words: acupuncture therapy, osteoarthritis, knee, subchondral bone, mechanical properties, bone morphogenetic protein 2, Smad1 protein

Chen Xilin, GUO Yan, LU Juan, QIN Luxue, HU Tingyao, ZENG Xin, WANG Xinyue, ZHANG Anran, ZHUANG Yuxin, ZHONG Honggang, GUO Changqing. Acupotomy ameliorates subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis by regulating bone morphogenetic protein 2-Smad1 pathway[J]. Journal of Traditional Chinese Medicine, 2023, 43(4): 734-743.

Figures/Tables 7

Table 1 Primers used in this study

Gene	Primer sequences
BMP2	Forward: 5'-CCCCTTAATTTGCAGAA ACAGTA-3'
	Reverse: 5'-GATCCCTGGTTTAGGAAAGAAGA-3'
Smad1	Forward: 5'-CCTTCCAGATGCCAGATGTT-3'
	Reverse: 5'-CCATCCACCAACACA CTCGT-3'
β-actin	Forward: 5'-TTGTCCCCCAACTTGAGATGTA-3'
	Reverse: 5'-GCACTTTTATTGAACTGGTCTCGT-3'

Table 2 Behavioral assessment of rabbits

Item	Group	n	Before intervention	After intervention
PROM	Control	9	141 (2)	141 (2.5)
	KOA	9	40 (9.5)^a	50 (18)^a
	Acupotomy	9	40.00 (14)^a	90 (27)^ac
	EA	9	38 (5)^a	98 (15)^ac
Lequesne MG	Control	9	0 (0)	0 (0)
	KOA	9	8 (1)^b	8 (0.5)^b
	Acupotomy	9	8 (0.5)^b	4 (1.5)^bd
	EA	9	7 (0.5)^b	4 (1)^bd

Figure 1 Cartilage degeneration under optical microscope A: the knee articular cartilage degeneration stained with Safranin O-Fast Green (× 200); A1: control group; A2: KOA group; A3: acupotomy group; A4: EA group; B: analysis of the Mankin score; C: analysis of the thickness of total articular cartilage. Data were mean ± standard deviation (n = 6). Control group: received no modeling and no intervention; KOA group: modeling for 6 weeks followed by no intervention; acupotomy group: modeling for 6 weeks followed by acupotomy intervention for 3 weeks; EA group: modeling for 6 weeks followed by electroacupuncture intervention for 3 weeks. TAC: total articular cartilage; KOA: knee osteoarthritis; EA: electroacupuncture. Compared with the control group, aP < 0.01, cP < 0.05; compared with the KOA group, bP < 0.01; compared with the EA group, dP < 0.01.

Figure 2 Subchondral bone remodeling under optical microscope A: the knee articular subchondral bone stained with Safranin O-Fast Green (×40); A1: control group; A2: KOA group; A3: acupotomy group; A4: EA group; B: the thickness of Subchondral bone plate; C: bone volume fraction. Data were mean ± standard deviation (n = 6). Control group: received no modeling and no intervention; KOA group: modeling for 6 weeks followed by no intervention; Acupotomy group: modeling for 6 weeks followed by acupotomy intervention for 3 weeks; EA group: modeling for 6 weeks followed by electroacupuncture intervention for 3 weeks. SBP: subchondral bone plate; BV/TV: bone volume/tissue volume; KOA: knee osteoarthritis; EA: electroacupuncture. Compared with the control group, aP < 0.01; compared with the KOA group, bP < 0.01; compared with the EA group, cP < 0.05, dP < 0.01.

Figure 3 Micro-CT scan and bone parameters analysis of subchondral bone A: Micro-CT scan of subchondral bone cylinder samples; A1-A4 shown transverse section; A5-A8 shown coronal section; A9-A12 shown 3D reconstruction; A1, A5, A9: control group; A2, A6, A10: KOA group; A3, A7, A11: acupotomy group; A4, A8, A12: EA group; B: trabecular thickness; C: trabecular separation/spacing; D: bone volume fraction. Data were expressed as mean ± standard deviation (n = 6). Control group: received no modeling and no intervention; KOA group: modeling for 6 weeks followed by no intervention; Acupotomy group: modeling for 6 weeks followed by acupotomy intervention for 3 weeks; EA group: modeling for 6 weeks followed by electroacupuncture intervention for 3 weeks. Tb. Th: the thickness of trabecular bone; Tb. Sp: the separation of trabecular bone; BV/TV: bone volume/tissue volume; KOA: knee osteoarthritis; EA: electroacupuncture. Compared with the control group, aP < 0.05, cP < 0.01; compared with the KOA group, bP < 0.01, dP < 0.05.

Table 3 Mechanical properties of subchondral bone (

\bar{x} \pm s

)

Group	n	Maximum load (N)	Elasticity modulus (MPa)
Control	6	117±21	265±42
KOA	6	33±10^a	100±24^a
Acupotomy	6	107±27^bc	226±45^bc
EA	6	76±32^ab	181±24^ab

Table 3 Mechanical properties of subchondral bone ( $\bar{x} \pm s$ )

Group	n	Maximum load (N)	Elasticity modulus (MPa)
Control	6	117±21	265±42
KOA	6	33±10^a	100±24^a
Acupotomy	6	107±27^bc	226±45^bc
EA	6	76±32^ab	181±24^ab

Figure 4 mRNA and protein expression of BMP2-Smad1 in subchondral bone A-B: mRNA expression of BMP2 and Smad1; C-E: Western blot of BMP2, p-Smad1, Smad1. Data were mean ± standard deviation (n = 6). Control group: received no modeling and no intervention; KOA group: modeling for 6 weeks followed by no intervention; Acupotomy group: modeling for 6 weeks followed by acupotomy intervention for 3 weeks; EA group: modeling for 6 weeks followed by electroacupuncture intervention for 3 weeks. BMP2: bone morphogenetic protein 2; KOA: knee osteoarthritis; EA: electroacupuncture. Compared with the control group, aP < 0.01, cP < 0.05; compared with the KOA group, bP < 0.01, dP < 0.05; compared with the EA group, eP < 0.01.

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