Behavior of cartilage-derived microtissue and ability of cartilage formation in three-dimensional dynamic and static culture conditions / 中国组织工程研究

BACKGROUND:Compared with traditional two-dimensional culture,three-dimensional microtissue culture can show greater advantages.However,more favorable cultivation methods in three-dimensional culture still need to be further explored. OBJECTIVE:To evaluate the cell behavior of microtissue and its ability to promote cartilage formation under two three-dimensional culture methods. METHODS:Cartilage-derived microcarriers were prepared by chemical decellularization and tissue crushing.DNA quantification and nuclear staining were used to verify the success of decellularization,and histological staining was used to observe the matrix retention before and after decellularization.The microcarriers were characterized by scanning electron microscopy and CCK-8 assay.Cartilage-derived microtissues were constructed by combining cartilage-derived microcarriers with human adipose mesenchymal stem cells through three-dimensional static culture and three-dimensional dynamic culture methods.The cell viability and chondrogenic ability of the two groups of microtissues were detected by scanning electron microscopy,live and dead staining,and RT-qPCR. RESULTS AND CONCLUSION:(1)Cartilage-derived microcarriers were successfully prepared.Compared with before decellularization,the DNA content significantly decreased after decellularization(P<0.001).Scanning electron microscope observation showed that the surface of the microcarrier was surrounded by collagen,maintaining the characteristics of the natural extracellular matrix of cartilage cells.CCK-8 assay indicated that microcarriers had no cytotoxicity and could promote cell proliferation.(2)Scanning electron microscopy and live and dead staining results showed that compared with the three-dimensional static group,the three-dimensional dynamic group had a more extended morphology of microtissue cells,and extensive connections between cells and cells,between cells and matrix,and between matrix.(3)The results of RT-qPCR showed that the expressions of SOX9,proteoglycan,and type Ⅱ collagen in microtissues of both groups were increased at 7 or 14 days.The relative expression levels of each gene in the three-dimensional dynamic group were significantly higher than those in the three-dimensional static group at 14 days(P<0.05).At 21 days,the three-dimensional static group had significantly higher gene expression compared with the three-diomensional dynamic group(P<0.001).(4)The results showed that compared with three-dimensional static culture microtissue,three-dimensional dynamic culture microtissue could achieve higher expression of chondrogen-related genes in a shorter time,showing better cell viability and chondrogenic ability.

In vitro construction of cartilage organoids based on extracellular matrix microcarriers of cartilage / 中华创伤杂志

Objective:To study the in vitro construction of functional and self-renewing cartilage organoids based on cartilage acellular extracellular matrix (ECM) microcarriers.Methods:Fresh porcine articular cartilage was taken. The merely crushed cartilage particles were set as natural cartilage group and ECM microcarriers of appropriate particle size, which were prepared by the acellular method of combining physical centrifugation and chemical extraction, were set as microcarrier group. Cartilage organoids were constructed by loading human umbilical cord mesenchymal stem cells (hUCMSCs) and human chondrocytes (hCho) with a ratio of 3∶1 with microcarriers through a rotating bioreactor. The organoids with different induction times were divided into 0-, 7-, 14-, and 21-day induction groups. The cell residues of the microcarrier group and natural cartilage group were evaluated by 4′, 6-diaminidine 2-phenylindole (DAPI) fluorescence staining and DNA quantitative analysis. The retention of microcarrier components was observed by Safranin O and toluidine blue stainnings, and the collagen and glycosaminoglycan (GAGs) levels in the microcarrier group and the natural cartilage group were determined by colorimetric method and dimethyl-methylene blue (DMMB) method. The microcarriers were further characterized by scanning electron microscopy and energy dispersive spectroscopy. The hUCMSCs cultured with Dulbecco′s Modified Eagle′s Medium (DMEM) supplemented with fetal bovine serum (FBS) in a volume fraction of 10% was used as the control group and the hUCMSCs cultured with the microcarrier extract was used as the experimental group. Subgroups of hUCMSCs cultured at 3 time points: 1, 3 and 5 days were set up in the two groups separately. Cell Counting Kit 8 (CCK-8) was used to detect the biocompatibility of the two groups. The cellular activity of the organoids of the 0-, 7-, 14-, and 21-day induction groups was detected by live/dead staining and the self-renewal ability of the cartilage organoids of the 14-day induced group was identified by Ki67 fluorescence staining. The organoids of the 7-, 14-, and 21-day induction groups were detected by RT-PCR in terms of the expression levels of chondrogenesis-related marker aggrecan (ACAN), type II collagen (COL2A1), SRY-related high mobility group-box gene-9 (SOX9), cartilage hypertrophy-and mineralization-related marker type I collagen (COL1A1), Runt-related transcription factor-2 (RUNX2), and osteocalcin (OCN). Colorimetric and DMMB assays were performed to determine the ability of organoids in the 0-, 7-, 14-, and 21-day induction groups to secrete collagen and GAGs.Results:The results of DAPI fluorescent staining showed that the natural cartilage group had a large number of nuclei while the microcarrier group hardly had any nuclei. The DNA content of the microcarrier group was (7.8±1.8)ng/mg, which was significantly lower than that of the natural cartilage group [(526.7±14.7)ng/mg] ( P<0.01). Saffranin O and toluidine blue staining showed that the microcarrier was dark- and uniform-colored and it kept a lot of cartilage ECM components. The collagen and GAGs contents of the microcarrier group were (252.9±1.4)μg/mg and (173.4±0.8)μg/mg, which were significantly lower than those of the natural cartilage group [(311.9±2.2)μg/mg and (241.3±0.7)μg/mg] ( P<0.01). Scanning electron microscopy showed that the surface of the microcarriers had uneven and interleaved collagen fiber network. The results of energy spectrum analysis showed that elements C, O and N were evenly distributed in the microcarriers, indicating that the composition of the microcarrier was uniform. The microcarrier had good biocompatibility and there was no statistical significance in the results of CCK-8 test between the control group and the experimental group after 1 and 3 days of culture ( P>0.05). After 5 days of culture, the A value of the experimental group was 0.53±0.02, which was better than that of the control group (0.44±0.03) ( P<0.05). In the 0-, 7-, 14-, and 21-day induction groups, hUCMSCs and hCho were attached to the surface of the microcarriers, with good cellular activity, and the live/death rates were (70.6±1.1)%, (80.5±0.6)%, (94.5±0.9)%, and (90.8±0.5)% respectively ( P<0.01). There were a large number of Ki67 positive cells in cartilage organoids. RT-PCR showed that the expression levels of ACAN, COL2A1, SOX9, COL1A1, RUNX2 and OCN were 1.00±0.09, 1.00±0.24, 1.00±0.18, 1.00±0.03, 1.00±0.06 and 1.00±0.13 respectively in the 7-day induction group; 4.16±0.28, 5.09±1.25, 5.65±1.05, 0.47±0.01, 1.68±0.02 and 0.21±0.06 respectively in the 14-day induction group; 13.42±0.92, 3.07±0.21, 1.84±1.08, 2.72±0.17, 2.91±0.18 and 3.32±1.20 respectively in the 21-day induction group. Compared with the 7-day induction group, the expression levels of ACAN, COL2A1, SOX9 and RUNX2 in the 14-day group were increased ( P<0.05), but COL1A1 expression level was decreased ( P<0.05), with no significant difference in OCN expression level ( P>0.05). Compared with the 7-day induction group, the expression levels of ACAN, COL1A1 and RUNX2 in the 21-day induction group were significantly increased ( P<0.01), with no significant differences in the expression levels of COL2A1, SOX9 and OCN ( P>0.05). Compared with the 14-day induction group, the expression levels of ACAN, COL1A1, RUNX2 and OCN in the 21-day group were increased ( P<0.05 or 0.01), with no significant difference in the expression level of COL2A1 ( P>0.05), but the expression level of SOX9 was decreased ( P<0.05). The contents of collagen in 0-, 7-, 14-and 21-day induction groups were (219.15±0.48)μg/mg, (264.07±1.58)μg/mg, (270.83±0.84)μg/mg and (280.01±0.48)μg/mg respectively. The GAGs contents were (171.18±1.09)μg/mg, (184.06±1.37)μg/mg, (241.08±0.84)μg/mg and (201.14±0.17)μg/mg respectively. Compared with the 0-day induction group, the contents of collagen and GAGs in 7-, 14-, and 21-day induction groups were significantly increased ( P<0.01), among which the content of collagen was the lowest in 7-day induction group ( P<0.01) but the highest in the 21-day induced group ( P<0.01); the content of GAGs was the lowest in the 7-day induced group ( P<0.01) but the highest in the 14-day induction group ( P<0.01). Conclusions:The microcarriers prepared by combining physical and chemical methods are decellularized successfully, with more matrix retention, uniform composition and on cytotoxicity. By loading microcarriers with hUCMSCs and hCho, cartilage organoids are successfully constructed in vitro, which are characterized by good cell activity, self-renewal ability, strong expression of genes related to chondrogenesis and secretion of collagen and GAGs. The cartilage organoids constructed at 14 days of induction have the best chondrogenic activity.

Expert consensus on the diagnosis and treatment of osteoporotic proximal humeral fracture with integrated traditional Chinese and Western medicine (version 2024) / 中华创伤杂志

Osteoporotic proximal humeral fracture (OPHF) is one of the common osteoporotic fractures in the aged, with an incidence only lower than vertebral compression fracture, hip fracture, and distal radius fracture. OPHF, secondary to osteoporosis and characterized by poor bone quality, comminuted fracture pattern, slow healing, and severely impaired shoulder joint function, poses a big challenge to the current clinical diagnosis and treatment. In the field of diagnosis, treatment, and rehabilitation of OPHF, traditional Chinese and Western medicine have accumulated rich experience and evidence from evidence-based medicine and achieved favorable outcomes. However, there is still a lack of guidance from a relevant consensus as to how to integrate the advantages of the two medical systems and achieve the integrated diagnosis and treatment. To promote the diagnosis and treatment of OPHF with integrated traditional Chinese and Western medicine, relevant experts from Orthopedic Expert Committee of Geriatric Branch of Chinese Association of Gerontology and Geriatrics, Youth Osteoporosis Group of Orthopedic Branch of Chinese Medical Association, Osteoporosis Group of Orthopedic Surgeon Branch of Chinese Medical Doctor Association, and Osteoporosis Committee of Shanghai Association of Integrated Traditional Chinese and Western Medicine have been organized to formulate Expert consensus on the diagnosis and treatment of osteoporotic proximal humeral fracture with integrated traditional Chinese and Western medicine ( version 2024) by searching related literatures and based on the evidences from evidence-based medicine. This consensus consists of 13 recommendations about the diagnosis, treatment and rehabilitation of OPHF with integrated traditional Chinese medicine and Western medicine, aimed at standardizing, systematizing, and personalizing the diagnosis and treatment of OPHF with integrated traditional Chinse and Western medicine to improve the patients ′ function.

Correlation analysis between arch index and foot kinematic parameters and their characteristics in stress fracture of lower extremity / 中华创伤杂志

Objective:To analyze the relationship between arch index and foot kinematic parameters and their characteristics in stress fracture of lower extremity.Methods:A case-control study was performed for 108 recruits selected from a certain army unit in 2019. Before training, the recruits′ foot print images were collected by the capacitive plantar pressure measurement system to calculate their arch indices. The kinematic characteristics of the foot were analyzed by the dynamic gait posture analysis system. Spearman rank correlation analysis between arch index and foot kinematic parameters including landing elevation angle, toe-off angle, landing speed, landing varus angle, valgus amplitude and landing valgus speed were performed. Throughout the training, orthopedic physicians followed up the recruits, among whom 10 were excluded due to other types of lower extremity injuries. The arch index and foot kinematic characteristics were analyzed and compared between the remained recruits with stress fracture of lower extremity (fracture group, n=10) and those without lower extremity injury (control group, n=79). Results:(1) For the recruits, the arch index was 0.21(0.12,0.25), with landing elevation angle for (17.31±4.02)°, toe-off angle for (63.90±5.63)°, landing speed for (176.85±24.39)°/s, landing varus angle for (13.64±4.44)°, valgus amplitude for (12.16±3.42)°, and landing valgus speed for 382.50(311.05,474.80)°/s. (2) The landing varus angle ( r=0.25, P<0.01) and valgus amplitude ( r=0.14, P<0.05) were positively related to the arch index. (3) The arch index, toe-off angle and landing valgus speed were 0.20(0.07,0.24), (61.59±5.51)° and 336.00(251.02,428.67)°/s in fracture group, significantly lower than 0.23(0.17,0.26), (64.79±4.79)° and 381.20(313.63,470.92)°/s in control group ( P<0.05 or 0.01). There were no significant differences in the landing elevation angle, landing speed, landing varus angle and valgus amplitude between the two groups (all P>0.05). Conclusions:The change of the arch index can affect the landing varus angle and valgus amplitude of the foot. Recruits who suffer from stress fracture of lower extremity have the characteristics of higher arch, lower toe-off angle and lower landing valgus speed.

Effects of bone-resorptive lesion on stress distribution of the femoral head and on progression in patients with osteonecrosis of the femoral head / 中华骨科杂志

Objective:To investigate effects of bone-resorptive lesion on stress distribution of femoral head and on progression in patients with osteonecrosis of the femoral head (ONFH).Methods:From April 2014 to September 2018, a total of 155 femoral heads from 94 patients diagnosed with ARCO stage II and III ONFH were retrospectively reviewed, including 77 males and 17 females with aged 39.90±10.45 years old (ranged from 18-64 years). The hips were divided into two groups according to whether there were bone-resorptive lesions. Further, we compared whether there was statistical difference between the two groups in staging. Then, a case of ARCO II hip joint without bone-resorptive lesion was selected from the included patients. Six femoral head with different diameters of spherical bone-resorptive lesion of 5 mm, 7 mm, 10 mm, 14 mm, 18 mm, and 23 mm were simulated. The influence of bone-resorptive lesion on the stress distribution of necrotic area and a spherical shell extending 1 mm radially around the bone-resorptive lesion was investigated by finite element method in slow walking conditions.Results:Of the 155 ONFH hips, 67 hips are complicated by bone-resorptive lesions, of which 17 were ARCO II, 50 were ARCO III. A total of 88 hips did not contain bone-resorptive lesions, of which 58 were ARCO II, ARCO III 30 cases. The proportion of ARCO stage II in the group with bone-resorptive lesions was significantly higher than that in the group without bone-resorptive lesions (χ 2=25.03, P=0.000). The finite element stress distribution cloud diagram showed that there was a stress concentration area around the bone-resorptive lesions. The maximum von Mises stress around bone-resorptive lesions in the models that contained a synthetic bone-resorptive lesions were significantly higher than those reported in the matched, non-synthetic bone-resorptive lesions finite element models ( t=3.139, P=0.026). The values for maximum von Mises stress around bone-resorptive lesions were 6.94±1.78 MPa and 5.01±0.35 MPa for the group with synthetic bone-resorptive lesions and the group non-synthetic bone-resorptive lesions, respectively. There was a positive correlation between the diameter of bone-resorptive lesions and the maximum and mean von Mises stress of necrotic areas as well as the maximum von Mises stress around bone-resorptive lesions. Conclusion:Bone-resorptive lesions can increase the maximum stress and average stress in the necrotic area. The larger the bone-resorptive lesion, the more the stress increases. There is a stress concentration area around the bone-resorptive lesions, which may accelerate the collapse of the femoral head.

Degradation of AZ31 magnesium alloysin vivo：micro-CT assessment / 中国组织工程研究

BACKGROUND:Magnesium can be degraded voluntarily in vivo, so a second surgery is avoided. However, its aloys have not been widely used in the clinical orthopedics because there is a lack of accurate and reliable methods to assess its degradationin vivo. OBJECTIVE:To explore the degradation of micro-arc-oxidized AZ31 magnesium aloy in the femoral condyle of rabbits based on micro-CT images and relative data. METHODS:Forty micro-arc-oxidized AZ31 magnesium aloys were implanted into the right femoral condyle of 40 New Zealand rabbits. Then 10 right femoral condyles were removed at 5, 10, 15 and 20 weeks after surgery, respectively, to quantitatively analyze and evaluate the degradation of AZ31 magnesium aloys by micro-CT images and relative data. RESULTS AND CONCLUSION:The surface of AZ31 aloys was corroded progressively with dark color and distorted appearance at 5-20 weeks post implantation. Micro-CT images showed that in the first 5 weeks, the degradation was inactive, and at the 10th week, it turned active; at the 15th week, the corrosion pits were obviously increased in number, and the corrosion area and corrosion speed were enlarged and fastened, respectively. Up to the 20th week, the aloy surfaces were ful of corrosion pits besides roughness and discontinuity. Relevant data analysis showed that the volume fraction of magnesium aloy was 98.6%, 97.1% and 86.4% at the 5th, 10th and 20th weeks after implantation, respectively, and it had a significant decrease from the 10th to 15th week and from the 15th to 20th week (P < 0.05). Within 15-20 weeks, the volume fraction of magnesium aloy was decreased by 6.5% that was the maximum volume reduction per unit cycle. With the progress of corrosion, the surface continuously became rough and vague, and its surface area was enlarged; the ratio of surface area to volume continuously increased, and there was a significant difference at 15 and 20 weeks (P < 0.05). Because of the increasing number of corrosion pits, the cross-sectional radius decreased, which was reflected by the trabecular thickness decreasing from 1.00 to 0.87 mm. From the view of the slope of curve, the trabecular thickness decreased most rapidly at 10-15 weeks. The mineral density of magnesium aloy continuously decreased from 649.302 to 356.445 mg/cm3 during the whole experiment period (P< 0.05). In addition, the micro-CT image density decreased from 679.710 to 644.947 mg/cm3, but there was no significant difference. To conclude, the degradation speed is peaked at 10-20 weeks after implantation, and the content of magnesium aloys decrease with degradation, but the magnesium density has no significant change.

NEL-like type 1 gene transfection for prevention of traumatic femoral head necrosis in rats / 中华创伤杂志

Objective To determine the effect of NEL-like type 1 gene (NELL-1) transfection in vivo in the repair of traumatic femoral head necrosis.Methods Twenty-four SD rats were randomly divided into three groups (8 rats per group) according to the lottery method,ie,sham group (served as normal control),NELL-1 treatment group (injected NELL-1 gene by recombinant adenovirus vectors around the hip one week after osteonecrosis model induced surgically) and placebo group (given an equal volume of saline solution at the same time after the induction of osteonecrosis).Femurs were taken from the animals 5 weeks after surgery.Gross observation was performed for morphology changes,X-ray assessment for femoral head height and length ratio (H/L),Micro-CT measure for bone parameters of femoral head including total volume (TV),bone volume (BV),total mineralized content (TMC),trabecular thickness (Tb.Th) and trabecular space (Tb.SP),and histological study for osteocytes,osteoblasts and osteoclasts.Results Preserved femoral head shape was noted in NELL-1 treatment group compared to the obvious flattening of the femoral head in placebo group.No heterotopic osteogenesis was observed in any group.Femoral head H/L ratio for 0.753 2 ± 0.040 2 in NELL-1 treatment group was higher than 0.598 4 ± 0.037 0 in placebo group (P ＜ 0.05),but lower than 0.920 2 ± 0.037 0 in sham group (P＜0.05).TV,BV,TMC and BMD between NELL-1 treatment and sham groups did not differ significantly (P ＞ 0.05),but all were increased compared to placebo group (P ＜ 0.05).There was no significant differences in Tb.Th and Tb.SP among three groups (P ＞ 0.05).Most osteocytes were alive in NELL-1 treatment group.More active osteoblasts and osteoclasts were noted in NELL-1 treatment group than those in placebo group.Conclusion NELL-1 gene transfection can preserve femoral head shape and bone content,promote osteoblast activity and neovascularization and hence is an effective treatment for rat traumatic osteonecrosis.However,the activity of osteoclasts is stimulated simultaneously.

Bioreactor cultivation and mechanical stimulation for regeneration of tissue-engineered cartilage / 中国组织工程研究

BACKGROUND:Cartilage tissue engineering has been widely used to achieve cartilage regeneration in vitro and repair cartilage defects. Tissue-engineered cartilage mainly consists of chondrocytes, cartilage scaffold and in vitro environment. OBJECTIVE:To mimic the environment of articular cartilage development in vivo, in order to increase the bionic features of tissue-engineered cartilage scaffold and effectiveness of cartilage repair. METHODS: Knee joint chondrocytes were isolated from New Zealand white rabbits, 2 months old, and expanded in vitro. The chondrocytes at passage 2 were seeded onto a scaffold of articular cartilage extracelular matrix in the concentration of 1×106/L to prepare cel-scaffold composites. Cel-scaffold composites were cultivated in an Instron bioreactor with mechanical compression (1 Hz, 3 hours per day, 10% compression) as experimental group for 7, 14, 24, 28 days or cultured staticaly for 1 day as control group. RESULTS AND CONCLUSION:Morphological observations demonstrated that the thickness, elastic modulus and maximum load of the composite in the experimental group were significantly higher than those in the control group, which were positively related to time (P < 0.05). Histological staining showed the proliferation of chondrocytes, formation of cartilage lacuna and synthesis of proteoglycan in the experimental group through hematoxylin-eosin staining and safranin-O staining, which were increased gradualy with mechanical stimulation time. These results were consistent with the findings of proteoglycan kit. Real-time quantitative PCR revealed that mRNA expressions of colagen type I and colagen type II were significantly higher in the experimental group than the control group (P < 0.05). The experimental group showed the highest mRNA expression of colagen type I and colagen type II at 21 and 28 days of mechanical stimulation, respectively (P < 0.05). With the mechanical stimulation of bioreactor, the cel-scaffold composite can produce more extracelular matrix, such as colagen and proteoglycan, strengthen the mechanical properties to be more coincident with thein vivo environment of cartilage development, and increase the bionic features. With the progress of tissue engineering, the clinical bioregeneration of damaged cartilage wil be achieved.

Inhibition of CCN1 siRNA on retinal endothelial cells / 中华实验眼科杂志

Background Cysteine-rich 61 (Cyr61)/CCN1 has been reported to stimulate retinal neovascularization (RNV) in retinopathy of prematurity (ROP).However, whether CCN1 small interfering RNA (CCN1 siRNA) can inhibit or cure ROP has not been extensively investigated.Objective This study was to investigate the regulation effect of CCN1 specific siRNA expression vector on retinal endothelial cells.Methods Rhesus choroid-retinal vascular endothelial cells (RF/6A) were cultured under the normoxic (normoxia control group) and hypoxic condition (1％ O2,5％ CO2 with 94％ N2) in vitro, and then lipofectamineTM 2000 (LF2000) vector plasmid with or without CCN1 siRNA was transiently transfected in the hypoxic-cultured cells as the CCN1 siRNA transfected group and hypoxic control group, respectively.Reverse transcription PCR was employed to detect the expression of CCN1 siRNA plasmid 24 hours after transfection.The vatality of the cells was assayed by cell counting kit-8 (CCK-8) 0,24,48,72 and 96 hours after cultured.Twenty-four hours after cultured,the apoptosis of the cells was evaluated by flow cytometry, and the expressions of CCN1 and vascular endothelial growth factor (VEGF) proteins were detected by immunofluorescence technique and Western blot assay.Results The expression band of CCN1 siRNA was detected in the cells 24 hours after transfection of CCN1 siRNA.CCK-8 assay showed that RF/6A cells were significantly increased over time, and the proliferating value (absorbancy) of the cells was significantly reduced in the CCN1 siRNA transfected group compared with in the normoxia control group and hypoxic control group (Fgroup =198.45, P＜0.05;Ftime =39.26, P＜ 0.05).The apoptosis rates of the cells were (68.9± 1.1) ％ , (18.9±1.3)％ and (39.6± 1.8)％ in the CCN1 siRNA transfected group, normoxia control group and hypoxic control group,and the apoptosis rates of the CCN1 siRNA transfected group were evidently higher than those of the normoxia control group and hypoxic control group (t =2.93 ,t=2.56 ,both at P＜0.05).CCN1 and VEGF proteins were weakly expressed in the normoxia control group and strongly expressed in the hypoxic control group,however,their expression intensity was evidently weakened in the CCN1 siRNA transfected group.The related expression levels of CCN1 and VEGF proteins in the CCN1 siRNA transfected group were significantly lower than those in the hypoxic control group (both at P＜0.05).Conclusions RNA interference targeting CCN1 can inhibit proliferation and promote apoptosis of RF/6A cells.CCN1 siRNA can arrest RNV probably by downregulating the expression levels of CCN1 and VEGF in the cells.

Variation of vascular endothelial growth factor in diabetics with asymp-tomatic and symptomatic peripheral artery disease / 中国现代医生

Objective To explore the serum level of vascular endothelial growth factor (VEGF) of the patients with asymptomatic and symptomatic peripheral artery disease (PAD) in diabetics for providing evidence for screening the patients with PAD early. Methods Selected the study population including diabetics without PAD(90 cases),diabetics with asymptomatic PAD patients(93 cases)and diabetics with symptomatic PAD patients(92 cases). The level of serum VEGF was measured using enzyme linked immunosorbent assay(ELISA) and analyzed in the three groups. The varia-tion of serum VEGF was further analyzed in the three groups excluding the diabetic retinopathy in the stratified analysis. Results The serum level of VEGF with asymptomatic and symptomatic PAD in diabetics were increased,compared with the diabetics without PAD (P<0.05). Excluding the patients with diabetic retinopathy, the serum level of VEGF of the patients with asymptomatic and symptomatic PAD in diabet ics was still increased, compared with the dia betics without PAD (P<0.05). The serum level of VEGF level was significantly positively associated with HbA1c in the pa-tients with asymptomatic and symptomatic PAD in diabetics (r=0.267, 0.352, P all<0.05). Conclusion The level of serum VEGF in the patients with asymptomatic and symptomatic PAD in diabetics had been significantly increased.

Mechanical tests on the reconstructed anterior cruciate ligament fixed with allogenetic cortical bone cross-pin on the femoral side / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Anterior cruciate ligament reconstruction (ACLR) has developed dramatically in the last century. Now, ACLR has become a reliable and productive procedure. Patients feel satisfied in >90% cases. The aim of this study was to evaluate the feasibility of allogenetic cortical bone cross-pin (ACBCP) used as a clinical fixation method in anterior cruciate ligament reconstruction on the femoral side based on biomechanical tests in vitro.</p><p><b>METHODS</b>The specimens were provided by the bone banks of the First Affiliated Hospital of People's Liberation Army of General Hospital from September 2011 to June 2012. Fresh deep frozen human allogenetic cortical bone was machined into cross-pins which is 4.0 mm in diameter and 75.0 mm in length. Biomechanical parameters compared with Rigidfix were collected while cross-pins were tested in double-shear test. The load-to-failure test and cycling test were carried out in a goat model to reconstruct anterior cruciate ligament with Achilles tendon autograft on the femoral side fixed by human 4.0 mm ACBCP and 3.3 mm Rigidfix served as control. Maximum failure load, yield load, and stiffness of fixation in single load-to-failure test were compared between the two groups. Cycle-specific stiffness and displacement at cycles 1, 30, 200, 400, and 1 000 were also compared in between.</p><p><b>RESULTS</b>In double-shear test both maximum failed load and yield load of 4.0 mm human ACBCP were (1 236.998±201.940) N. Maximum failed load and yield load of Rigidfix were (807.929±110.511) N and (592.483±58.821) N. The differences of maximum failed load and yield load were significant between ACBCP and Rigidfix, P < 0.05. The shear strength of ACBCP and Rigidfix were (49.243±8.039) MPa and (34.637±3.439) MPa, respectively, P < 0.05. In the load-to-failure test ex vivo, yield load and maximum failed load of ACBCP fixation complexity ((867.104±132.856)N, (1 032.243±196.281) N) were higher than those of Rigidfix ((640.935±42.836) N, (800.568±64.890) N, P < 0.05). However, stiffness did not differ significantly between ACBCP group ((247.116±31.897)N/mm) and Rigidfix group ((220.413±51.332) N/mm, P > 0.05). In the cycling test, the cycle-specific stiffness and displacement at cycles 1, 30, 200, 400, and 1 000 did not differ significantly between the ACBCP group and Rigidfix group, P > 0.05.</p><p><b>CONCLUSIONS</b>Allogenetic cortical bone cross-pin possesses satisfactory biomechanical profile which is safe for ACLR and suitable for an aggressive rehabilitation program. Animal and clinical tests should be recommended before clinical use to secure the ACBCP could successfully substituted by host new bone in vivo.</p>

Fabrication and characterization of a novel cartilage acellular matrix/chitosan hybrid scaffold for cartilage tissue engineering / 中华骨科杂志

Objective To explore the feasibility of fabricating a novel cartilage acellular matrix/chitosan hybrid scaffold for cartilage tissue engineering. Methods Human cartilage microfilaments about 100 nm-5 μm were prepared after pulverization and made into 1% suspension after decellularization. The suspension was mixed with 2% chitosan acetic acid solution, and then hybrid scaffolds were fabricated using a simple freeze-drying method. The scaffolds were cross-linked and were investigated by histological staining,SEM observation, porosity measurement, water absorption rate, biomechanical properties, and biocompatibility analysis. MTT test was also done to assess the cytotoxicity of scaffold leaching liquor. Canine chondrocytes were isolated and seeded into the scaffold. Cell proliferation and differentiation were analyzed using inverted microscope and SEM. Results The histological staining showed no chondrocyte fragments remained in the scaffolds, and anti-col Ⅱ immunohistochemistry staining were positive. SEM observation show the scaffold has good pore interconnectivity with pore diameter (136.2±34.9) μm, 81.4%±3.5% porosity and 1525.7%±129.3% water absorption rate. The longitudinal elastic modulus of the scaffold was (1.940±0.335) MPa. MTT test showed that the scaffold leaching liquor did not exert any cytotoxic effect on BMSCs. Inverted microscope and SEM micrographs indicatod that cells covered the scaffolds uniformly, and majority of the cells showed the round or elliptic morphology with much matrix secretion. Conclusion Novel cartilage acellular matrix/chitosan hybrid scaffold had similar extracellular matrix as cartilage, good pore diameter and porosity,appropriate biomechanical character, non-toxicity and good biocompatibility, which make it a suitable candidate as an alternative cell-carrier for cartilage tissue engineering.

Repair of large articular high-load-bearing osteochondral defects in canine hip joint using tissue-engineered osteochondral constructs / 中华骨科杂志

Objective To investigate the effects of the novel scaffold on repairing large,high-loadbearing osteochondral defects of femoral head in a canine model.Methods The biphasic scaffolds were fabricated using cartilage extracellular matrix (ECM)-derived scaffold (cartilage layer) and acellular bone matrix (bone layer) by phase separation technique.Articular high-load-bearing osteochondral defects with a diameter of 11-mm and the depth of 10-mm were created in femoral heads.The defects were treated with constructs of a biphasic scaffold seeded with chondrogenically induced bone marrow-derived mesenehymal stem cells (BMSCs).The outcomes were evaluated for gross morphology,histological,biomechanical and micro-CT analysis at the third and sixth month after implantation.Results The gross and X-ray results showed femoral head slightly collapsed at the third month and severely collapse at the sixth month.Histological analysis showed cartilage defects were repaired with fibrous tissue or fibrocartilage with severe osteoarthritis and the varied degrees of the collapse of femoral heads were presented.Micro-CT showed that the values of bone volume fraction in defect area were always lower than those of the normal area in the femoral heads.Biomechanical analysis showed rigidity of the subchondral bone in defect area was significantly lower than that in normal area in the femoral heads at the sixth month.Conclusion The ECM-derived,integrated biphasic scaffold seeded with chondrogenically induced BMSCs could not successfully repair the large high-load-bearing osteochondral defects of the femoral head.

In vitro short term cartilage tissue engineering with oriented cartilage extracellular matrix scaffolds and bone marrow mesenchymal stem cells / 中华骨科杂志

Objective To fabricate cartilage extracellular matrix (ECM) oriented scaffolds and investigate the attachment, proliferation, distribution and orientation of bone marrow mesenchymal stem cells (BMSCs) cultured within the scaffolds in vitro. Methods Cartilage slices were shattered in sterile phosphate-buffered saline (PBS) and the suspension were differentially centrifugated untill the micro- fiber of the cartilage extracellular matrix was disassociated from the residue cartilage fragments. At last the supernatant were centrifugated, the precipitation were collected and were made into 2%-3% suspension. Using unidirectional solidification as a freezing process and freeze-dried method, the cartilage extracellular matrix derived oriented scaffolds was fabricated. The scaffolds were then cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution. By light microscope and scan electron microscope (SEM) observation, histological staining, and biomechanical test, the traits of scaffolds were studied. After being labelled with PKH26 fluorescent dye, rabbit BMSCs were seeded onto the scaffolds. The attachment, proliferation and differentiation of the cells were analyzed using inverted fluorescent microscope. Results The histological staining showed that toluidine blue, safranin O, alcian blue and anti-collagen Ⅱ immunohistochemistry staining of the scaffolds were positive. A perpendicular pore-channel structures which has a diameter of 100 μm were verified by light microscope and SEM analysis. The cell-free scaffolds showed the compression moduli were (2.02±0.02) MPa in the mechanical testing. Inverted fluorescent microscope showed that most of the cells attached to the scaffold. Cells were found to be widely distributed within the scaffold, which acted as a columnar arrangement. The formation of a surface cells layer was found on the surface of the scaffolds which resembled natural cartilage. Coclusion The cartilage extracellular matrix derived oriented scaffolds have promising biological, structural, and mechanical properties.

Stiffness changes in short-segment spine after destabilizing by using the Distraction Reduction Fixation System / 中国组织工程研究

BACKGROUND:Self-made Distraction Reduction Fixation System is characterized by Distraction Reduction Fixation functions,which can completely fuse to the design of steel plate,and by no need to provide many surgical instruments,by reduction of bleeding and trauma.OBJECTIVE:To test changes in the stiffness of porcine short spinal segments in integrity and following destabilizing and internal fixation of the Distraction Reduction Fixation System.DESIGN,TIME AND SETTING:The control observation experiment was performed at the Laboratory of Biodynamics,Institute of Onhopedics,General Hospital of Chinese PLA from December 2004 to December 2005.MATERIALS:Twelve fresh porcine lumbar spines(T11-S2 segments)were bought from a market.Upper and lower segments were crossed and inserted with Kirschner wire to make porcine spine samples of the L1-7 segments.METHODS:Stiffness of intact samples was recorded during forward-bending,backward-bending,lateral-bending and axial rotation.Intervertebral discs,facet ioints,anterior and posterior longitudinal ligaments were removed to loose the spine.Stifrness of each motion was measured.Stiffness tests were repeated again after internal fixation on short segments using Distraction Reduction Fixation System.MAIN OUTCOME MEASURES:Stiffness in the intact,unstable and internal fixed spines during each motion.RESULTS:The stiffness iu unstable porcine spine were significantly lower than those in intact porcine spine(P<0.05),but the stiffness after internal fixation was significantly higher than in unstable porcine spine(P<0.05).No significant difference was detected in stifiness during forward-bending,lateral-bending and axial rotation(P>0.05)except that it was significantly higher during backward.bending after internal fixation compared with the intaet spine(P<0.05).CONCLUSION:Biodynamics confirms that the stiffness in short-segment fixation porcine after using Distraction Reduction Fixation System is markedly increased than in unstable porcine spine,except during backward-bending,which is comparable with that in intact porcine spine.

Comparison of morphology and mechanical properties between vitrified rabbit femoral arteries and cryopreserved rabbit femoral arteries / 中国组织工程研究

BACKGROUND: An effective preservation method must preserve the integrity of tissue structure. OBJECTIVE: To compare the effects of vitrification and cryopreservation method on the artery morphology and mechanical properties. DESIGN, TIME AND SETTING: The randomized controlled animal experiment was done at the Department of Orthopedics, General Hospital of Chinese PLA, between September 2001 and August 2004. MATERIALS: Eighteen New Zealand rabbits were randomly divided into vitrified artery group, cryopreserved artery group and fresh artery group, with 6 rabbits in each group. METHODS: Femoral arteries were removed from rabbits and put in balanced solution. Arteries in the vitrified artery group were immersed in the 25%, 50% and 100% gradient vitrified solution at 4 ℃ and then were put in liquid nitrogen. Arteries in the cryopreserved artery group were cooled from normal temperature to 0, -20, -70 ℃, and balanced for 60 minutes, then were put in liquid nitrogen. Samples were preserved in liquid nitrogen for more than 14 days. MAIN OUTCOME MEASURES: The morphology changes of preserved arteries observed through naked eye and microscope; hysteresis loop; stress relaxation; breaking strength. RESULTS: Artery structures were all preserved well in the three groups, the integrity rate of vitrified artery group was 91.67%, which was significantly better than 54.17% of cryopreserved artery group (P 0.05). CONCLUSION: There influences of vitrification and cryoprsesrvation on the artery morphology and mechanical properties were not significant, while arteries preserved by vitrification had less tissue ruptures, so vitrification is suitable for preserving long vessels.

Experimental study of recovery force of surface-modified TiNi memory alloy rod / 生物医学工程学杂志

The recovery force of Ti-Nb coated and uncoated TiNi shape memory alloy rods was investigated. The rods were 6.0 mm, 6.5 mm and 7.0 mm in diameter respectively. The mean transition temperature was 33.0 degrees C. The rods were stored at -18 degrees C and pre-bent with a three-point bending fixture, the span was 20. 0 centimeters and the deflections were 5.0 mm, 10.0 mm, 15.0 mm and 20.0 mm, respectively. The rods were then heated in a constant temperature saline solution chamber. The experimental temperature was 37.0 C and 50.0 C respectively. The recovery force was measured in a constant displacement mode on biomaterial test machine. The results showed that the recovery force of the memory alloy rod increased with increasing recovery temperature, rod diameter and deformation of both Ti-Nb coated and uncoated surface. The recovery force of Ti-Nb coated rods of 6.0 and 6.5 millimeter in diameter was lower than the uncoated rods in the same diameter. However, the recovery force of 7.0-mm-diameter rods showed no significant difference between coated and uncoated surface.

Dynamic observation of ossification process in a rat femur distraction model by living small animal Micro-CT / 中国矫形外科杂志

[Objective]In order to dynamically observe the ossification process by living small animal Micro-CT in a rat femur distraction osteogenesis (DO) model,the author applied a special designed external fixiator system. [Methods]A femur DO model was made on 12 SD rats.After 7 days of latency,the femurs were distracted at a speed of 0.25 mm every 12 hours for 14 days.At the first day of consolidation period,the steel external fixiators were substituted by radio-transparent polymer splint material made fixiators.At 7 and 21 days of consolidation period,two randomly selected rats were sacrificed for histological examination.The other 8 rats were selected for X-ray and living Micro-CT examination at 0,7,21,35 days of consolidation period.All animals were sacrificed at 35 days of consolidation period,5 for biomechanical test,3 for histological examination.[Results]There was no significant new bone formed in distracted zone at latency and distraction periods.The volume of distracted zone increased little in the following 5 weeks of consolidation period.Bone volume,bone mineral content,bone volume fraction,and the number of trabeculae all increased over the consolidation period,while mineral content of mineralized tissue and thickness of traleculae did not increased until 3 weeks after consolidation began.At final observation (56 days post operation),there still exist un-mineralized cartilage tissues in the central of distracted zone,and the distracted femurs had lower biomechanical property when compared with corresponding normal femurs.[Conclusion]1) Radio-transparent polymer splint is a satisfactory substitute of steel external fixator.2) Micro-CT is a useful tool for dynamical observing and evaluating bone formation status in DO model.3) The increasing of bone volume began at the end of distraction,lasting at least 5 weeks,while the remodeling of new formed bone began 3 weeks after distraction ended.The increasing and remodeling of new formed bone was not concord.This result indicates that,in order to obtain proper results,the corresponding interference should be taken at different time points.

Micro-CT evaluation of femoral head samples' trabecular structure with femoral neck fracture / 中国矫形外科杂志

0.05).[Conclusion]The values of three-dimensional parameters in old femoral neck fracture patient are different with that of normal persons in the loading region of the femoral head although there is no difference in BMD values between them.This may provide new evidence to predict osteoporotic femoral neck fracture.

Role of exogenous epidermal growth factor in nerve regeneration after terminolateral neurorrhaphy / 中国组织工程研究

BACKGROUND: It is proved that nerve regeneration induced by terminolateral neurorrhaphy(TLN) is not as active as that induced by end-to-end suture. Exogenous epidermal growth factor(EGF) increases the opportunity of neuron survival in vitro and promotes nerve regeneration. Whether it can increase nerve regeneration after terminolateral neurorrhaphy deserves further study.OBJECTIVE: To evaluate the effect of exogenous EGF in promoting nerve regeneration after terminolateral neurorrhaphy.DESIGN: A randomized controlled trial.SETTING: Orthopedic Institute of Chinese PLA General Hospital.PARTICIPANTS: The trial was conducted in the Orthopedic Institute of Chinese PLA General Hospital from September 2001 to February 2002. A total of 32 male Wistar rats, weighting 200- 250 g, were randomized to control group and EGF group with 16 rats in each group.METHODS: The right peroneal nerve was transected and an epineural window of 1 mm was created on the neighboring tibial nerve. The distal end of the transected peroneal nerve was sutured to the windowed tibial nerve by means of end-to-side attachment. Each rat in EGF group received injection of 0. 1 mL/d EGF diluted with normal saline at 2 g/L for two weeks while each in control group received injection of normal saline (0. 1 mL/d) at the distal site of the transected peroneal nerve for two weeks. Histological, morphological and electrophysiological examinations were performed 4 and 8 weeks after operation.MAIN OUTCOME MEASURES: The regeneration rate of myelinated nerve, motor nerve conduction velocity and ultrastructural changes of the two groups.rate of myelinated nerve fibers: 4 and 8 weeks after operation, it was better in EGF group[ (52.42 ± 1.45)% and(61.41 ± 1.54)% ] than that in control nerve conduction velocity: 4 and 8 weeks after operation it was obviously greater in EGF group[ (30. 33 ±0. 88)m/s and(34. 36 ± 1.09)m/s] than that in conObservation of ultrastructure: The number of myelinated nerve fibers, and the thickness and maturation degree of myelin sheath were significantly better than those in control group.CONCLUSION: Exogenous EGF can promote nerve regeneration, increase nerve conduction velocity after terminolateral neurorrhaphy.