The effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament reconstruction: An animal study.

OBJECTIVES: To compare the effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament (ACL) reconstruction animal model. METHODS: Anterior cruciate ligament reconstruction using the plantaris tendon as graft material was performed on both knees of 24 rabbits (48 knees) to mimic ACL reconstruction by two different suspensory fixation devices for graft fixation. For the adjustable fixation device model (Socket group; group S), a 5 mm deep socket was created in the lateral femoral condyle (LFC) of the right knee. For the fixed-loop model (Tunnel group; group T), a femoral tunnel penetrating the LFC was created in the left knee. Animals were sacrificed at four and eight weeks after surgery for histological evaluation and biomechanical testing. RESULTS: Histologically, both groups showed a mixture of direct and indirect healing patterns at four weeks, whereas only indirect healing patterns were observed in both groups at eight weeks. No significant histological differences were seen between the two groups at four and eight weeks in the roof zone (four weeks, S: mean 4.8 sd 1.7, T: mean 4.5 sd 0.5, p = 0.14; eight weeks, S: mean 5.8 sd 0.8, T: mean 4.8 sd 1.8, p = 0.88, Mann-Whitney U test) or side zone (four weeks, S: mean 5.0 sd 1.2, T: mean 4.8 sd 0.4, p = 0.43; eight weeks, S: mean 5.3 sd 0.8,T: mean 5.5 sd 0.8, p = 0.61, Mann-Whitney U test) . Similarly, no significant difference was seen in the maximum failure load between group S and group T at four (15.6 sd 9.0N and 13.1 sd 5.6N) or eight weeks (12.6 sd 3.6N and 17.1 sd 6.4N, respectively). CONCLUSION: Regardless of bone tunnel configuration, tendon-bone healing after ACL reconstruction primarily occurred through indirect healing. No significant histological or mechanical differences were observed between adjustable and fixed-loop femoral cortical suspension methods.Cite this article: Y. Sato, R. Akagi, Y. Akatsu, Y. Matsuura, S. Takahashi, S. Yamaguchi, T. Enomoto, R. Nakagawa, H. Hoshi, T. Sasaki, S. Kimura, Y. Ogawa, A. Sadamasu, S. Ohtori, T. Sasho. The effect of femoral bone tunnel configuration on tendon-bone healing in an anterior cruciate ligament reconstruction: An animal study. Bone Joint Res 2018;7:327-335. DOI: 10.1302/2046-3758.75.BJR-2017-0238.R2.

The effect of systemic administration of G-CSF on a full-thickness cartilage defect in a rabbit model MSC proliferation as presumed mechanism: G-CSF for cartilage repair.

OBJECTIVES: The aim of this study was to investigate the effect of granulocyte-colony stimulating factor (G-CSF) on mesenchymal stem cell (MSC) proliferation in vitro and to determine whether pre-microfracture systemic administration of G-CSF (a bone marrow stimulant) could improve the quality of repaired tissue of a full-thickness cartilage defect in a rabbit model. METHODS: MSCs from rabbits were cultured in a control medium and medium with G-CSF (low-dose: 4 µg, high-dose: 40 µg). At one, three, and five days after culturing, cells were counted. Differential potential of cultured cells were examined by stimulating them with a osteogenic, adipogenic and chondrogenic medium.A total of 30 rabbits were divided into three groups. The low-dose group (n = 10) received 10 µg/kg of G-CSF daily, the high-dose group (n = 10) received 50 µg/kg daily by subcutaneous injection for three days prior to creating cartilage defects. The control group (n = 10) was administered saline for three days. At 48 hours after the first injection, a 5.2 mm diameter cylindrical osteochondral defect was created in the femoral trochlea. At four and 12 weeks post-operatively, repaired tissue was evaluated macroscopically and microscopically. RESULTS: The cell count in the low-dose G-CSF medium was significantly higher than that in the control medium. The differentiation potential of MSCs was preserved after culturing them with G-CSF.Macroscopically, defects were filled and surfaces were smoother in the G-CSF groups than in the control group at four weeks. At 12 weeks, the quality of repaired cartilage improved further, and defects were almost completely filled in all groups. Microscopically, at four weeks, defects were partially filled with hyaline-like cartilage in the G-CSF groups. At 12 weeks, defects were repaired with hyaline-like cartilage in all groups. CONCLUSIONS: G-CSF promoted proliferation of MSCs in vitro. The systemic administration of G-CSF promoted the repair of damaged cartilage possibly through increasing the number of MSCs in a rabbit model.Cite this article: T. Sasaki, R. Akagi, Y. Akatsu, T. Fukawa, H. Hoshi, Y. Yamamoto, T. Enomoto, Y. Sato, R. Nakagawa, K. Takahashi, S. Yamaguchi, T. Sasho. The effect of systemic administration of G-CSF on a full-thickness cartilage defect in a rabbit model MSC proliferation as presumed mechanism: G-CSF for cartilage repair. Bone Joint Res 2017;6:123-131. DOI: 10.1302/2046-3758.63.BJR-2016-0083.

Dysregulated circadian rhythm pathway in human osteoarthritis: NR1D1 and BMAL1 suppression alters TGF-ß signaling in chondrocytes.

OBJECTIVES: Circadian rhythm (CR) was identified by RNA sequencing as the most dysregulated pathway in human osteoarthritis (OA) in articular cartilage. This study examined circadian rhythmicity in cultured chondrocytes and the role of the CR genes NR1D1 and BMAL1 in regulating chondrocyte functions. METHODS: RNA was extracted from normal and OA-affected human knee cartilage (n = 14 each). Expression levels of NR1D1 and BMAL1 mRNA and protein were assessed by quantitative PCR and immunohistochemistry. Human chondrocytes were synchronized and harvested at regular intervals to examine circadian rhythmicity in RNA and protein expression. Chondrocytes were treated with small interfering RNA (siRNA) for NR1D1 or BMAL1, followed by RNA sequencing and analysis of the effects on the transforming growth factor beta (TGF-ß) pathway. RESULTS: NR1D1 and BMAL1 mRNA and protein levels were significantly reduced in OA compared to normal cartilage. In cultured human chondrocytes, a clear circadian rhythmicity was observed for NR1D1 and BMAL1. Increased BMAL1 expression was observed after knocking down NR1D1, and decreased NR1D1 levels were observed after knocking down BMAL1. Sequencing of RNA from chondrocytes treated with NR1D1 or BMAL1 siRNA identified 330 and 68 significantly different genes, respectively, and this predominantly affected the TGF-ß signaling pathway. CONCLUSIONS: The CR pathway is dysregulated in OA cartilage. Interference with circadian rhythmicity in cultured chondrocytes affects TGF-ß signaling, which is a central pathway in cartilage homeostasis.

Utility of T2 mapping and dGEMRIC for evaluation of cartilage repair after allograft chondrocyte implantation in a rabbit model.

OBJECTIVE: To investigate the effectiveness of quantitative Magnetic resonance imaging (MRI) for evaluating the quality of cartilage repair over time following allograft chondrocyte implantation using a three-dimensional scaffold for osteochondral lesions. DESIGN: Thirty knees from 15 rabbits were analyzed. An osteochondral defect (diameter, 4 mm; depth, 1 mm) was created on the patellar groove of the femur in both legs. The defects were filled with a chondrocyte-seeded scaffold in the right knee and an empty scaffold in the left knee. Five rabbits each were euthanized at 4, 8, and 12 weeks and their knees were examined via macroscopic inspection, histological and biochemical analysis, and quantitative MRI (T2 mapping and dGEMRIC) to assess the state of tissue repair following allograft chondrocyte implantation with a three-dimensional scaffold for osteochondral lesions. RESULTS: Comparatively good regenerative cartilage was observed both macroscopically and histologically. In both chondrocyte-seeded and control knees, the T2 values of repair tissues were highest at 4 weeks and showed a tendency to decrease with time. ΔR1 values of dGEMRIC also tended to decrease with time in both groups, and the mean ΔR1 was significantly lower in the CS-scaffold group than in the control group at all time points. ΔR1 = 1/r (R1post - R1pre), where r is the relaxivity of Gd-DTPA(2-), R1 = 1/T1 (longitudinal relaxation time). CONCLUSION: T2 mapping and dGEMRIC were both effective for evaluating tissue repair after allograft chondrocyte implantation. ΔR1 values of dGEMRIC represented good correlation with histologically and biochemically even at early stages after the implantation.

Hidden osteophyte formation on plain X-ray is the predictive factor for development of knee osteoarthritis after 48 months--data from the Osteoarthritis Initiative.

OBJECTIVE: To examine whether the detection of osteophytes anywhere in the knee could serve as a pre-radiographic biomarker for osteoarthritis (OA) development. METHODS: Baseline magnetic resonance imaging (MRIs) of 132 participants in the Osteoarthritis Initiative (OAI) were studied. Based on radiographs, 66 knees were assessed as osteoarthritis-free (no-osteoarthritis [NOA], or Kellgren/Lawrence [K/L] severity grade 0/1 both at baseline and 48 months), and another 66 knees were assessed as having radiographic OA changes (pre-radiographic osteoarthritis [PROA], or with K/L grade 0/1 at baseline and grade ≥ 2 at 48 months). Using baseline MRI data, we examined eight sites of osteophyte formation: the medial and lateral femoral condyle (MFC and LFC, respectively); medial and lateral tibial plateau (MTP and LTP, respectively); medial and lateral facets of the patellofemoral joint (PM and PL, respectively); tibial spine (TS); and femoral intercondylar notch (IC). Knee joint osteophyte size was assessed via the 8-point marginal osteophytes item of the whole-organ magnetic resonance imaging score (WORMS). The frequencies and distributions of osteophytes were compared between groups. RESULTS: Mild-size osteophytes (defined as score ≥ 2) were observed more frequently at the MFC (P = 0.00278), MTP (P = 0.0046), TS (P = 0.0146), PM (P < 0.0001), PL (P = 0.0012), and IC (P < 0.0001) in PROA knees than in NOA knees. Moderate-size osteophytes (defined as score ≥ 4) were more frequently observed in PROA knees than in NOA knees only at the IC (P < 0.0001). CONCLUSION: Knees with osteophyte formation at the IC, even those of K/L severity grade 0/1, are at risk for the development of radiographic OA by 48 months.

Preventive effects of hyaluronan from deterioration of gait parameters in surgically induced mice osteoarthritic knee model.

OBJECTIVE: Osteoarthritis (OA) leads to pain and loss of function in affected joints. Gait disturbance results from these symptoms of OA, and gait analysis can be important to evaluate the progression of OA. The purpose of this study was to analyze gait pattern in a rodent model of OA and to assess the effects of intra-articular injection of hyaluronan (IAI-HA) by gait analysis, along with histological evaluation. DESIGN: OA was induced by destabilization of the medial meniscus (DMM) of C57BL/6 mice. IAI-HA started 3 weeks after DMM surgery. Mice were allocated to three groups and were given either 800-kDa HA (800-HA), 6000-kDa HA (6000-HA) or saline. We compared these three groups with a sham group by gait analysis using CatWalk. Histological evaluation was performed to assess articular cartilage changes in the knee joints. RESULTS: Mice injected with 800-HA or 6000-HA showed gait patterns similar to that of the sham mice, while the saline-injected group showed gait disturbances 12 and 16 weeks after DMM surgery. Histological changes in articular cartilage were similar among the 800-HA, 6000-HA and saline-treated groups, demonstrating OA progression throughout the experimental time points. Positive gait-related effects of IAI-HA might occur by its pain relieving effect and/or by preventing contracture. CONCLUSION: IAI-HA prevented gait disturbances in the DMM model, but did not prevent histological changes associated with OA progression.

Attenuation of natural killer cell activity during 2-h exercise in individuals with spinal cord injuries.

DESIGN: Non-randomized study. OBJECTIVE: To determine natural killer cell cytotoxic activity (NKCA) to 2-h arm ergometer exercise in persons with spinal cord injuries (SCI) and the underlying mechanism of such response. SETTING: University of Occupational and Environmental Health, Japan. METHODS: We examined NKCA response to 2-h arm crank ergometer exercise at 60% of maximum oxygen consumption (VO(2max)) in SCI and able-bodied persons. NKCA and plasma concentrations of prostaglandin E(2) (PGE(2)), adrenaline and cortisol were measured before, during and immediately after the exercise. The study included seven subjects with SCI between Th11 and L4 and six able-bodied persons. RESULTS: NKCA in able-bodied subjects increased (P<0.05) at 60 min of exercise and immediately after the exercise, and remained elevated up to 2 h after exercise. However, NKCA in SCI decreased (P<0.05) immediately after exercise but recovered at 2 h after exercise. Plasma adrenaline in both groups increased significantly (P<0.05) immediately after exercise and returned to baseline level 2 h after the exercise. Plasma cortisol in both groups remained constant throughout the study. In SCI, PGE(2) significantly increased immediately after 2 h exercise and returned to the baseline level 2 h after exercise; however, it remained unchanged during the test in able-bodied subjects. CONCLUSION: Our results suggested that increase of PGE(2) in SCI partially contributes to NKCA.

Isokinetic strength of elbow extensor muscles correlates with race time in wheelchair half marathon racers.

It has been reported that grasping power and isometric muscle strength of elbow extensors in fine wheelchair racers were greater than the poor racers in the wheelchair full marathon. The purpose of the present study was to investigate the relationship between muscle strength of elbow extensors and flexors and the race time in wheelchair half marathon race. Four wheelchair half marathon racers who completed the half marathon division of the 16th Oita International Wheelchair Marathon (OIWM) in 1996 participated in the present study. The day before the race, all subjects reported to the training room and the isokinetic muscle strength of elbow extensor and flexor muscle groups was measured at angular speeds of 60 degrees, 120 degrees and 240 degrees/sec using the isokinetic dynamometer. There was a significant correlation between race time and isokinetic muscle strength of the elbow extensors at 60 degrees, 120 degrees and 240 degrees/sec, but not flexor muscles. Our findings suggest that increased muscle strength of elbow extensors may improve the race time in wheelchair half marathon race.

Complementary improvement of the method for determining cholinergic activities in the small intestine and its application to experiments in vivo.

The concentration of acetylcholine (ACh) in rat jejunum that had been homogenized with an ultra-high-speed homogenizer (Biotron) was significantly higher than that in jejunum homogenized with a glass homogenizer. Rats were injected once or repeatedly for 10 days with a muscarinic agonist, pilocarpine (1 mg/kg), or a muscarinic antagonist, scopolamine (5 mg/kg). Animals were killed 20 min or 24 hr after single or consecutive injections, respectively, for determinations of cholinergic activities in the jejunum. Single treatment: Pilocarpine did not cause significant changes in the level of ACh, the activity of acetylcholinesterase (AChE), the binding of [3H]quinuclidinyl benzilate (QNB) or the contractile responses to ACh. Scopolamine reduced the level of ACh and binding of [3H]QNB without inducing significant changes in the activity of AChE and the contractile response. Consecutive treatment: Pilocarpine reduced the binding of [3H]QNB by changing the value of Bmax and reduced the contractile response without affecting the level of ACh or the activity of AChE. Scopolamine increased the binding of [3H]QNB without any effects on the level of ACh, the activity of AChE or the contractile response. In summary, it is possible to determine the level of ACh in a tissue as hard as intestine by homogenization with a Biotron and to assess the cholinergic situation in the intestine of animals that have been poisoned with various agents by estimating cholinergic activities.

Effects of single or repeated administration of a carbamate, propoxur, and an organophosphate, DDVP, on jejunal cholinergic activities and contractile responses in rats.

Wistar rats were injected once or repeatedly for 10 days with dichlorvos (DDVP, 5 mg kg-1), propoxur (10 mg kg-1), oxotremorine (0.1 mg kg-1) or atropine (5 mg kg-1). Animals were killed 20 min or 24 h after single or consecutive injections, respectively, for determinations of cholinergic activities and contractile responses to acetylcholine (ACh) of the jejunum. Single treatments: while DDVP and propoxur decreased acetylcholinesterase (AChE) activity, oxotremorine and atropine did not. Although DDVP, propoxur and oxotremorine increased levels of ACh, atropine decreased them. Contractile responses to ACh were enhanced by DDVP and reduced by oxotremorine and atropine. The Bmax value of binding of [3H]quinuclidinyl benzylate (QNB) to muscarinic ACh receptors was decreased by atropine. Consecutive treatments: DDVP and oxotremorine decreased AChE activity markedly and slightly, respectively. Although DDVP and oxotremorine increased levels of ACh, propoxur decreased them. Without affecting the contractile responses, DDVP caused a reduction and propoxur and atropine caused an increase in the Bmax value for binding of [3H]QNB. Both the contractile responses and the value of Bmax for binding of [3H]-QNB were decreased by oxotremorine. In summary, propoxur and DDVP showed similar effects mainly through their anticholinesterase properties in the case of single injection, but DDVP had similar effects to those of oxotremorine and propoxur had similar effects to those of atropine in the case of repeated injection.