OBJECTIVE To explore a new surgical approach to repair facial paralysis in late stage, using regional transposition of pedicled sternocleidomastoid muscle for the dynamic reanimation of the paralyzed face. METHODS Seven cases with facial paralysis in late stage from December 1999 were treated and followed up for 10 months before clinical evaluation. In all of the cases, the sternal and clavicular branches of the sternocleidomastoid muscle were both elevated from their bony attachments, with the mastoid insertion left in situ as the pedicle for blood supply and accessory nerve maintained in it. The muscle strips were transposed and sutured to the orbicularis oris around the mouth corner on the paralyzed side. RESULTS Static asymmetry of nose and oral commissure on the paralyzed side were corrected immediately after operation, and the movement of the oral commissure recovered one week after operation. Symmetric smiling was observed in one month and all of the oral movements recovered in 10 months postoperatively. CONCLUSION The new approach to repair facial paralysis in late stage by regional transposition of pedicled sternocleidomastoid muscle is effective in restoration of both static and dynamic symmetry of nose and mouth, and in recovery of the facial expression and the oral commissure.
OBJECTIVE: To study the relationship between intracellular actin and scar contracture. METHODS: Fibroblasts from 10 cases of hypertrophic scar and 5 cases of keloid were cultured in vitro. Total actin, filamentous actin(F actin), globular actin (G actin) and the ratio of F to G actin(F/G) were measured by densitometry after differential extraction and separation by polyacrylamide gel electrophoresis in the presence of sodium sulfate. RESULTS: Total actin, F actin, G actin and F/G in hypertrophic scar fibroblasts were 2.38 ng/10(4) cells, 0.98 ng/10(4) cells, 1.42 ng/10(4) cells and 0.68 respectively, while in keloid fibroblasts were 1.68 ng/10(4) cells. 0.46 ng/10(4) cells, 1.26 ng/10(4) cells, and 0.36 respectively. There was significant differences between two tissues fibroblasts in the items of total actin, F actin, G actin, and F/G (P lt; 0.01), while no significant difference in G actin (P gt; 0.05). CONCLUSION: Total intracellular actin, F actin, and F/G may play an important role in the scar contracture. The hypertrophic scar and keloid can be distinguished by the contents of total intracellular actin, F actin and F/G.
Objective To investigate the feasibil ity of replacing urinary epithel ial cells with oral mucosa cell to reconstruct tissue engineered urethra by being seeded on bladder acellular matrix graft (BAMG). Methods Eighteen male New Zealand rabbits, aged 10 weeks, weighing 0.3-0.5 kg, were used in this study. Oral mucosa cell of 12 rabbits were isolated and seeded onto a culture dish with a feeder layer of 3T3 and a culture dish without 3T3, respectively. The morphologic change and growth condition of oral mucosa cells were observed by inverted phase contrast microscope after 2 days of seeding. The quantity of oral mucosa cells was counted using cell counting meter; the cell growth curve was drawn and the immunofluorescence staining with broad-spectrum keratin antibody was carried out. The bladders taken from the rest 6 rabbits were decelluled to make BAMG and the tissue of 1 cm × 1 cm was randomly selected to observe the effect of acellularization. The second passage oral mucosa cells cultured with 3T3 were appl ied to steril ized BAMG to obtain a issueengineered mucosa. The tissue-engineered mucosa was assessed using HE staining and scanning electron microscope after being cultured for 1 week. Results Oral mucosa cells seeded onto a feeder layer of 3T3 could be passaged for 7 or 8 generations with homogeneous forms and full function. Oral mucosa cells cultured without 3T3 could only be subcultured for 2 generations before aging and had multiple shapes and different sizes. Oral mucosa cells cultured by the two methods both started logarithmic growth on the 8th day and reached the peak value on the 14th day, which was indicated by the cell growth curve. However, more cells could be obtained through oral mucosa cells cultured with 3T3 than those cultured without 3T3. Oral mucosa cells manifestated green colour fluorescence cultured with or without 3T3. After the cells were removed, the BAMG presented as a porous membrane. The HE staining showed that the effect of acellularization was good and there were no cells at BAMG. The second passage oral mucosa cells cultured with 3T3 were expanded and seeded onto steril ized BAMG to obtain a tissue-engineered mucosa. Good compatibil ity of the compound graft was assessed using HE staining and scanning electron microscope. HE staining and scanning electron microscope showed that oral mucosa cells had good biocompatibil ity with BAMG after the tissue engineered mucosa was cultured for 1 week. Conclusion Oral mucosa cells of rabbit can be cultured in vitro and attain magnitude quantities. Oral mucosa cell also have good biocompatibil ity with BAMG and the compound graft could be a new material for urethral reconstruction.
ObjectiveTo investigate the anatomic features of the perforating branch flap of the medial vastus muscle, so as to provide a new perforating branch flap for repairing the soft tissue defect. MethodsSix fresh donated lower limb specimens underwent an intra-arterial injection of a lead oxide and lactoprene preparation. The integument of the thigh was dissected to observe the origin, course, size, and location of the perforating branch of the medial vastus muscle by angiography and photography. Based on the anatomic study, the free perforating branch flaps of the medial vastus muscle (14 cm × 6 cm to 20 cm × 5 cm) were used to repair skin and soft tissue defects (8 cm × 6 cm to 12 cm × 8 cm) of the feet in 4 patients between June 2009 and August 2011. ResultsThe artery of the medial vastus was sent out constantly from the femoral artery, and then descended in the vastus muscle to lateral patella where it anastomosed with the terminal branches of lateral femoral circumflex artery to form prepatellar vascular network. The artery of the medial vastus sent out 3-5 musculocutaneous perforating branches into the deep fascia and then extended superficially to the overlying skin. Four flaps survived after surgery; wounds at the donor site and recipient site healed by first intention. After follow-up of 6-12 months, the flaps had good appearance and texture. All ankles had normal movement range of plantarflexion and dorsiflexion. ConclusionThe free perforating branch flaps of the medial vastus muscle can be harvested easily, and have the advantage of good texture and abundant donor site.
Objective Calcium phosphate bioceramics has a broad appl ication prospect because of good biocompatibil ity, but porous scaffolds with complex shape can not be prepared by the traditional methods. To fabricate porous calcium phosphate ceramics by rapid prototyping and to investigate the in vitro osteogenic activities. Methods The porous calcium phosphate ceramics was fabricated by rapid prototyping. The bone marrow mesenchymal stem cells (BMSCs)were isolated from bone marrow of Beagle canine, and the 3rd passage BMSCs were seeded onto the porous ceramics. The cell/ceramics composite cultured in osteogenic medium were taken as the experimental group (group A) and the cell/ceramics composite cultured in growth medium were taken as the control group (group B). Meanwhile, the cells seeded on the culture plate were cultured in osteogenic medium or growth medium respectively as positive control (group C) or negative control (group D). After 1, 3, and 7 days of culture, the cell prol iferation and osteogenic differentiation on the porous ceramics were evaluated by DNA quantitative analysis, histochemical staining and alkal ine phosphatase (ALP) activity. After DiO fluorescent dye, the cell adhesion, growth, and prol iferation on the porous ceramics were also observed by confocal laser scanning microscope (CLSM). Results DNA quantitative analysis results showed that the number of BMSCs in all groups increased continuously with time. Plateau phase was not obvious in groups A and B, but it was clearly observed in groups C and D. The CLSM observation indicated that the activity of BMSCs was good and the cells spread extensively, showing good adhesion and prol iferation on the porous calcium phosphate ceramics prepared by rapid prototyping. ALP quantitative analysis results showed that the stain of cells on the ceramics became deeper and deeper with time in groups A and B, the staining degree in group A were ber than that in group B. There was no significant difference in the change of the ALP activity among 4 groups at the first 3 days (P gt; 0.05); the ALP activity increased obviously in 4 groups at 7 days, group A was significantly higher than other groups (P lt; 0.05) and groups C, D were significantly higher than group D (P lt; 0.05). Conclusion The porous calcium phosphate ceramics has good cytocompatibil ity and the designed pores are favorable for cell ingrowth. The porous ceramicsfabricated by rapid prototyping has prominent osteogenic differentiation activity and can be used as a choice of scaffolds for bone tissue engineering.
Objective To establ ish a two-dimensional biological printing technique of hBMSCs so as to control the cell transfer process and keep cell viabil ity after printing. Methods Bone marrow (5 mL) was obtained from healthy volunteer. The hBMSCs were regularly subcultured to harvest cells at passage 2, which were adjusted to the single cell suspensionat a density of 1 × 106/mL. The experiment was divided into 3 groups: printing group 1 in which cells underwent propidium iodide (PI) fluorescent label ing, then were transferred by rapid prototype biological printer (interval in x-axis 300 μm, interval in y-axis 1 500 μm), and laser scanning confocal microscope was appl ied to observe cell fluorescence; printing group 2 in which cells received no PI label ing and were cultured for 2 hours after transfer, Live/Dead viabil ity Kit was adopted to detect cell viabil ity and laser scanning confocal microscope was appl ied to observe cell fluorescence; half of the cells in printing group receiving no Live/Dead viabil ity Kit detection were cultured for 7 days, then inverted microscope was used to observe cell morphology, routine culture was conducted after the adherence of cells, the growth condition of cells was observed dynamically; control group in which steps were the same as the printing group 2 except that cell suspension received no printing. Results Laser scanning confocal microscope observation on the cells in printing group 1 revealed the “cell ink droplets” were distributed regularly and evenly in the two-dimensional layer and each contained 15-35 cells, meeting the requirement of designing two-dimensional cell printing. The cells in printing group 2 went through cell viabil ity test, laser scanning confocal microscope observation showed the fluorescence of cells 30 minutes after cell incubation. There was no significant difference between the control group and the printing groups in terms of cell viabil ity. The printed cells presented normal adherence, good morphology and good growth state 7 days after routine culture. Conclusion Biological printing technique can real ize the oriented, quantificational and regulardistribution of hBMSCs in the two-dimensional plane and lays the foundation for the construction of three-dimensional cellprinting or even organ printing system.
To study the feasibil ity of human adipose derived stem cells (ADSCs) in monolayer culture induced into smooth muscle cells in vitro as seeding cells in vascular tissue engineering. Methods The mononuclear cells in human adipose were separated by collagenase treatment and seeded on culture dishes with the density of 5 × 105/cm2. Cellswere cultured in M-199 plus 10% FBS. When reaching confluence, the cells were subcultured by 0.1% trypsin and 0.02%EDTA treatment, PDGF-BB (50 ng/mL) and TGF-β1 (5 ng/mL) were added at the passage 1 to enhance the smooth muscle cells’ phenotype. Cells were cultured under the inducing medium for 14 days. The morphology of induced cells was observed under the microscope. Cellular immunofluorescence and RT-PCR were used to determine the expression of smooth muscle cell markers of the post-induced cells. Flow cytometry (FACs) was used to examine the positive rate of induced team. Results Cocultured in M-199 media including TGF-β1 and PDGF-BB, the prol iferating capabil ity of the induced cells was significantly downregulated compared with the uninduced cells(P lt; 0.01). The induced cells exhibited “Hill and Valley” morphology, while the uninduced cells were similar to ADSCs of P0 which had the fibroblast-l ike morphology. The results of immunofluorescence indicated that the induced cells expressed smooth muscle (SM) cell- specific markers including α-smooth muscle actin (α-SMA), SM-myosin heavy chain (SM-MHC) and Calponin. The results of RT-PCR revealed that the induced cells also expressed α-SMA, SM-MHC, Calponin and SM-22α.The positive rates of α-SMA, SM-MHC and Calponin in FACs were 3.26% ± 1.31%, 3.55% ± 1.6% and 4.02% ± 1.81%, respectively, before the cells were induced. However, 14 days after the cell induction, the positive rates were 48.13% ± 8.31%, 45.33% ± 10.68% and 39.13% ± 9.42%, respectively. The positive rates in induced cells were remarkably higher than those in uninduced cells(P lt; 0.01). Conclusion The human ADSCs can be induced to express vascular smooth muscle markers, and they are a new potential source of vascular tissue engineering.
ObjectiveTo study the effect of chemical extraction of allogeneic tendon and allogeneic chondrocytes for reconstruction of anterior labrum of shoulder joint in rabbits.MethodsThe body weight of 45 adult New Zealand white rabbits ranged from 2.5 to 3.0 kg. The Achilles tendons of 15 rabbits were taken and the allogeneic tendons were prepared by chemical extraction with antigen inactivation. The extracted tendons were compared with untreated tendons by HE and Masson stainings. Chondrocytes were isolated and cultured by trypsin method and identified by immunohistochemical staining of collagen type Ⅱ. The remaining 30 rabbits were used to prepare the model of anterior labrum defect of shoulder joint. After the allogeneic tendon was transplanted to the damaged labrum, the rabbits was randomly divided into two groups (15 in each group). In group A, the allogeneic chondrocytes were injected into the joint immediately after transplantation, while in group B, no treatment was made. At 4, 6, and 8 weeks after operation, 5 transplanted tendons of each group were taken. After general observation, HE staining was used to observe the number of nuclei, Masson staining was used to observe the expression of collagen fibers in muscle fiber tissues, and AB staining was used to detect the glycosaminoglycan level after transplantation, to evaluate the cell growth in the tissues of the two groups of allogeneic tendon.ResultsBy HE and Masson stainings, the allogeneic tendon antigen prepared by chemical extraction method was inactivated and the fibrous tissue structure was intact; collagen type Ⅱ immunohisto-chemistry staining showed that the cultured cells were chondrocytes. After tendon transplantation, the content of glycosaminoglycan in group A was significantly higher than that in group B (P<0.05). At 6 weeks after operation, HE staining showed that the nuclear in tendon tissue of group A was significantly more than that of group B (t=20.043, P=0.000). Masson staining showed that the number of nuclei in tendon tissue of group A was significantly increased, the muscle fibers and collagen fibers were interlaced, the tissue structure was more compact, and the tendon tissue was mainly blue stained; while the number of nuclei in group B was less, mainly collagen fibers of the original graft.ConclusionThe allogeneic tendon inactivated by chemical extraction can be used to reconstruct the defect of anterior labrum of shoulder joint in rabbits, and the combination of allogeneic chondrocytes can promote the healing of tendon transplantation.
Objective To explore the effectiveness of two-stage operation on free latissimus dorsi myocutaneous flap transplantation and skull contour reconstruction in the treatment of head titanium mesh exposure complicated with soft tissue infection. Methods Between January 2015 and December 2021, 13 patients with head titanium mesh exposure complicated with soft tissue infection were admitted. There were 9 males and 4 females with a mean age of 42.9 years (range, 23-64 years). The duration of titanium mesh exposure was 22-609 days (median, 102 days). The wound site located at the frontal part in 3 cases, the parietal part in 1 case, the occipital part in 2 cases, the frontal-parietal part in 1 case, the temporal-parietal part in 4 cases, and the frontotemporal part in 2 cases. The titanium mesh had been taken out in 5 patients before admission, leaving skull defect and shape collapse, with signs of infection. The bacterial culture was positive in 7 cases and negative in 6 cases. The imaging examination revealed that the size of the skull defect ranged from 6 cm×5 cm to 21 cm×17 cm and the scalp defect ranged from 1 cm×1 cm to 15 cm×10 cm. The soft tissue infection did not reach dura in 5 cases, reached dura in 6 cases, and reached frontal sinus in 2 cases. The two-stage surgical protocol was used in all patients. In the first-stage operation, the latissimus dorsi myocutaneous flap was designed to repair the skull and scalp defects after removing the titanium mesh and thorough debridement. The size of muscle flap ranged from 13.5 cm×4.0 cm to 21.0 cm×17.0 cm, and the skin flap ranged from 7.0 cm×4.0 cm to 15.0 cm×10.0 cm. After the flap survived and stabilized, the second-stage operation was performed. The titanium mesh was implanted to reconstruct the skull contour. The size of titanium mesh ranged from 7.0 cm×6.0 cm to 21.5 cm×17.5 cm. The interval between the first- and second-stage operations was 3.7-17.8 months, with an average of 11.4 months. The survival of the skin flap, the appearance of the head, and the presence of re-exposed titanium mesh and infection were observed after operation. Results At the first-stage operation, venous embolism occurred in 1 case, and no obvious abnormality was observed after treatment. All the flaps survived and the incisions healed by first intention. Besides, the incisions of the second-stage operation healed by first intention. All patients were followed up 1-96 months (median, 14 months). During follow-up, no exposure to titanium mesh, infection, or other complications occurred. The appearance satisfaction rate of the patients was 92.31% (11/13). There was no significant difference in the skull contour between the affected side and the healthy side in all patients. Conclusion For the head titanium mesh exposure with soft tissue infection, the application of two-stage operation on free latissimus dorsi myocutaneous flap transplantation and skull contour reconstruction can reduce the risks of implant exposure and infection again by increasing the thickness of the scalp and blood supply, filling the wound cavity, and obtain good effectiveness.