Callus Research Articles

Antioxidant Activity, Polyphenolic Content, and FT-NIR Analysis of Different Aspilia africana Medicinal Plant Tissues.

Aspilia africana has been used for generations to treat many diseases in Africa. Its biological activities, including antioxidant and anti-inflammatory potential, are attributed to a number of secondary metabolites, including alkaloids and polyphenolics. The antioxidant activities of A. africana callus (CA), juvenile in vitro leaf (IL) and root (IR), ex vitro root (SR) and leaf (SL), and wild leaf (WL) dried samples were assessed based on their diphenylpicrylhydrazyl (DPPH) free radical scavenging abilities. The total phenolic and flavonoid content of different plant samples was compared. Further, high-pressure liquid chromatography (HPLC) was used to quantitatively determine chlorogenic acid content in the A. africana plant samples. Fourier transform near-infrared (FT-NIR) analysis was also carried out to compare the antioxidant phytochemical content in the A. africana plant tissues. Among the samples, IR, with the highest total phenolic content (167.84 ± 1.057 mg GAE/g), total flavonoid content (135.06 ± 0.786 mg RUE/g), and chlorogenic acid (5.23 ± 0.298 mg/g) content, had the most potent antioxidant activity (IC50 = 27.25 ± 5.028 μg/mL), followed by WL. The lowest polyphenolic content and antioxidant activity were observed in SR. The antioxidant activities of A. africana tissues were positively correlated with the total phenolic and flavonoid content in the samples. The differences in antioxidant activities of A. africana tissues could be attributed to the difference in their polyphenolic content. Our study reports, for the first time, the antioxidant activities of A. africana callus and roots (in vitro and ex vitro). The A. africana samples IR, CA, and WL could be valuable natural sources of antioxidants that could be further exploited for the development of useful pharmaceutical products.

Memecik Zeytin Çeliklerinin Köklenmesi Üzerine Etilenin Etkisi

Bu çalışmada, Memecik zeytin çeliklerinin köklenmesi üzerine etilenin etkisinin belirlenmesi amaçlanmıştır. Çalışmada 10 farklı bitki büyüme düzenleyici madde kombinasyonu uygulamasında her uygulamada 3 tekerrür, her tekerrürde 30’ar adet olmak üzere toplam 900 adet zeytin yarı odun çeliği kullanılmıştır. Çalışma sonucunda en yüksek köklenme oranı %44.4 ile 0 etilen+6000 ppm IBA uygulamasından elde edilirken, bunu %42.2 ile 0 etilen+8000 ppm IBA uygulaması izlemiştir. En düşük köklenme oranı %2.2 ile 0 etilen+0 ppm IBA (kontrol) ve 150 etilen+4000 ppm IBA (dikimden 10 gün sonra) uygulamalarında gözlenmiştir. Çeliklerin dikimden itibaren farklı günlerde yapılan bitki büyümeyi düzenleyici maddeleri uygulamalarında en yüksek kallus oranı (% 44.7) 150 etilen+4000 ppm IBA (20.gün)’da, en fazla kök sayısı (13.3 adet) 0 etilen+6000 ppm IBA’da, en fazla kök uzunluğu (7.0 cm) 50 etilen+4000 ppm IBA (10.gün)’da ve en fazla kök çapı (3.0 mm) 150 etilen+4000 ppm IBA (10.gün)’da elde edilmiştir. Sürgün uzunluğu, çapı ve sayısı bakımından uygulamalar arasındaki farklılık önemli bulunmamıştır. Artan etilen dozları Memecik yarı odun çeliklerinin köklenmesini azalttığı için 50 ppm’den daha düşük etilen dozlarının denenmesi tavsiye edilmektedir.

Effect of Elicitors treated callus of Baliospermum montanum (Willd.) Muell. Arg. on cell cycle arrest and apoptosis in HEP-2 and PC-3 cells

Effect of Elicitors treated callus of Baliospermum montanum (Willd.) Muell. Arg. on cell cycle arrest and apoptosis in HEP-2 and PC-3 cells

Development of metabolic engineering approaches to regulate the content of total phenolics, antiradical activity and organic acids in callus cultures of the highbush blueberry (Vaccinium corymbosum L.)

Development of metabolic engineering approaches to regulate the content of total phenolics, antiradical activity and organic acids in callus cultures of the highbush blueberry (Vaccinium corymbosum L.)

Initiation of callus from different genotypes of Sorghum bicolor L. Moench

The study aimed to examine three genotypes of sorghum for their response in tissue culture using Murashige and Skoog (MS) medium supplemented with auxins (2,4-dichlorophenoxy acetic acid (2,4-D), naphthalene acetic acid (NAA)) and cytokinins (Kinetin, 6-benzyle amino purine (BAP)) at different concentrations. The cultures were initiated from different explants (seed, embryo and hypocotyl). The response of explants varied with the genotype. Callus culture were initiated successfully on MS medium supplemented with 2 mg/l 2,4-D and 0.5 mg/l Kinetin from G4 (L58) seedling and embryo explants. G2 (94) hypocotyl explants gave callus on media supplemented with 2,4-D only. G5 (2) explants failed to initiate callus but bulging from the embryo explants was observed on MS medium supplemented with 2 mg/l 2,4-D and 0.5 mg/l Kinetin. The initiated calluses differed morphologically and in their growth rates. Key words: Sorghum bicolor, callus formation, embryo, Kinetin, 2,4-dichlorophenoxy acetic acid (2,4-D).

The first report: The effect of garlic extract on rooting of cuttings of some ornamental plants and fruit trees

One of the problems with the use of cuttings, particularly woody and semi-woody ones, is to root them for propagation of various plant species. The use of rooting hormones involves a high cost of foreign exchange and creates many environmental problems. Garlic extract has a lot of antimicrobial properties and can prevent the release of microorganisms in the culture medium of plants. In this study, garlic extract at three concentrations (0, 25, and 50 g/L) was investigated on the rooting of three different species include easy-to-root cuttings (rose, wild privet, and poplar), moderate-root cuttings (sycamore, berry, and grape), and hard-to-root cuttings (apple, sour cherry, and cherry) in three applications of the culture substrate, on the cuttings, and in the callus stage. Results showed that garlic extract can be used at a concentration of 25 g/L at the callus formation stage to improve the quantity and quality of grape cuttings in terms of shooting and rooting. The quantity and quality of shoots and roots for wild privet cuttings can be improved with garlic extract at 25 g/L and be used on the cuttings. Garlic extract at concentrations of 50 and 25 g/L can be used in the callus stage to enhance the quantity and quality of sycamore and berry cuttings, respectively. No positive effects were observed in the other plants, or positive effects were found only either on shooting or on rooting traits.

Expression of gus and gfp genes in amphidiploid spelt wheat (Triticum spelta L.) after Agrobacterium-mediated transformation

Мета. Дослідити експресію генів gus та gfp в калюсних експлантах амфідиплоїдної пшениці спельти ( Triticum spelta L.) після Agrobacterium -опосередкованої генетичної трансформації. Методи. Для трансформації було обрано сорт пшениці спельти озимої ‘Європа’. Як експланти використовували калюси, отримані зі зрілих зародків. Прекультивацію калюсів здійснювали на живильному середовищі МС (Мурасіге–Скуга), доповненому 2 мг/л 2,4-Д (2,4-дихлорфеноксиоцтова кислота) та 10 мг/л нітратом срібла. Для генетичної трансформації використовували Agrobacterium tumefaciens Conn., штам GV3101, та генетичну конструкцію з репортерними генами gus (ген бета-глюкуронідази (-glucuronidase)) та gfp (ген зеленого флюоресцентного білка (Green Fluorescent Protein, GFP)). Калюси трансформували шляхом інокуляції з агробактеріями та вакуумною інфільтрацією. Далі їх ко-культивували на середовищі МС із 2 мг/л 2,4-Д та 10 мг/л AgNO 3 , але без антибіотиків. Експресію гена gus перевіряли за допомогою гістохімічного, а гена gfp – візуального аналізу (флуоресценція білка GFP в УФ світлі). Рівні експресії генів gfp та gus оцінювали за допомогою програмного забезпечення ImajeJ. Інтеграцію генів gfp та gus в геном спельти перевіряли методом ПЛР . Результати. Генетична трансформація калюсних експлан­тів спельти шляхом їхньої інокуляції в живильному середовищі з агробактеріями та вакуумною інфільтрацією відбувалась з різною частотою. Рівень експресії гена gus за вакуумної інфільтрації становив 4,66 ± 0,74%, за інокуляції – 4,00 ± 0,91%; а гена gfp за вакуумної інфільтрації – 3,66 ± 0,74%, за інокуляції – 4,66 ± 1,39%. Рівень експресії гена gfp був вищим у разі використання інокуляції з агробактеріями, а гена gus – при ва­ку­умній інфільтрації. За допомогою ПЛР-аналізу було під­тверджено інтеграцію генів gfp та gus в геном калюсів спельти. Довжина ПЛР продукту із праймерами до гена gus становила 240 п. н., а до гена gfp – 717 п. н. Висновки. Використання методів вакуумної інфільтрації та інокуляції для генетичної трансформації спельти дали різні результати. Частота генетичної трансформації коливалась від 3,66 до 4,66%. Agrobacterium -опосередкована генетична трансформація амфідиплоїдної пшениці спельти дозволяє дослідити експресію репортерних генів gus та gfp за використання калюсних експлантів, отриманих із зрілих зародків.

Closed percutaneous reduction by leverage and Kirschner wire fixation for treatment of irreducible phalangeal neck fractures in children

Objective To investigate the clinical efficacy of closed percutaneous reduction by leverage and Kirschner wire fixation in the treatment of irreducible phalangeal neck fractures in children. Methods From January 2016 to March 2019, 12 children with irreducible phalangeal neck fractures ware treated. The time from injury to operation was 1 to 46 days, including 5 cases of old fracture. Closed Kirschner wire puncture was used to clear callus and closed percutaneous reduction by leverage and Kirschner wire fixation were performed. The fracture healing time of the children was followed up after operation. Total active motion (TAM) system was used to evaluate the recovery of finger function. Results All the operations were completed successfully, and no infection, skin necrosis and fracture malunion occurred. All the children were follow-up for 6 to 36 months, with an average of 15.3 months. The fracture healing time was (4.2±0.2) weeks. The Kirschner wire was removed and functional exercises were performed 4 weeks after operation. The finger joint function was evaluated as excellent according to TAM system. Conclusion Closed percutaneous reduction by leverage and Kirschner wire fixation in the treatment of irreducible phalangeal neck fractures in children has the advantages of simple operation, less trauma and definite curative effect. It is necessary to strictly grasp the operative indications for old fracture. Key words: Finger phalanges; Fractures,bone; Fracture fixation; Kirschner wire

Research progress in effect of alendronate on healing of osteoporotic fracture

Fracture healing is a complex physiological process involving osteoblasts, osteoclasts and other cells and molecules. Typical fracture healing can be divided into four stages: inflammatory response, soft callus formation, hard callus formation, and bone remodeling. Osteoclasts play a leading role in hard callus formation and bone remodeling. Alendronate can inhibit osteoclast activity and bone loss in patients with osteoporosis, but it may also inhibit fracture healing. Therefore, whether alendronate can be used after osteoporotic fracture is controversial. In recent years, it has been found that alendronate can not affect the fracture healing, but also reduce the risk of secondary fracture and improve the prognosis of patients. In this article, the mechanism of alendronate and its effect on osteoporotic fracture healing by systemic and local use are reviewed, which can provide a reference for clinical selection of therapeutic drugs. Key words: Osteoporotic fractures; Fracture healing; Alendronate

Treatment of closed calcaneal fractures by open reduction and internal fixation with bone plate through a small posterior heel plus tarsal canal incision

Objective To report the treatment effects of open reduction and internal fixation with bone plate through a small posterior heel plus tarsal canal incision on closed calcaneal fractures. Methods A retrospective study was done of the 20 patients (25 feet) who had been treated at Ward One, Department of Orthopaedics, People's Hospital of Yunfu from February 2016 to February of 2019 for closed calcaneal fractures by open reduction and internal fixation with bone plate through a small posterior heel plus tarsal canal incision. They were 16 males and 4 females, aged from 16 to 60 years. According to the Sanders classification, there were 3 cases of type Ⅱ, 15 cases of type Ⅲ and 2 cases of type Ⅳ. Their fractures were reduced by traction, extruding, prying and direct visualization through the tarsal canal window; the bone plates were inserted through a small incision at the back of the heel and fixated by screws. Postoperative observation was done to address fracture healing, and length, width, height, Bohler angle and Gissane angle of the affected calcaneus, as well as functional recovery of the ankle-hindfoot by the American Orthopaedic Foot and Ankle Society (AOFAS) evaluation. Results The operation time for a single foot ranged from 45 min to 70 min, averaging 64.5 min; the intraoperative fluoroscopy for a single foot ranged from 3 times to 6 times, averaging 4.5 times. Local skin necrosis of about 0.5 cm×0.3 cm appeared in one foot after operation but responded to dressing change. No other wound complications occurred. Their follow up was carried out for 6 to 36 months (average, 17.3 months). The fractures healed well with well-shaped bony callus and flat articular surface after 4 to 6 months. The length (80.5 mm±4.2 mm), width (44.8 mm±5.2 mm), height (44.4 mm±3.0 mm), Bohler angle (25.0°±5.1°) and Gissane angle (113.8°±8.6°) of the calcaneus at the last follow up were significantly improved than the preoperative values (79.4 mm ± 4.5 mm, 50.5 mm ± 6.3 mm, 40.0 mm±4.4 mm, 12.0°±13.8° and 107.0°±13.3°) (all P<0.05). By the AOFAS ankle-hindfoot scale, functional recovery of the foot was excellent in 20, good in 3 and fair in 2 cases, giving an excellent to good rate of 92%. Conclusion In the treatment of closed calcaneal fractures, open reduction and internal fixation with bone plate through a small posterior heel plus tarsal canal incision may lead to fine outcomes due to its advantages of small incision and fine fracture reduction. Key words: Calcaneus; Fractures, bone; Fracture fixation, internal; Tarsal Canal; Small incision

Experimental study of the time effect of controlled micromovement on the influence of the fracture healing

Objective To explore the influence and mechanism of time effect of the controlled micromovement on fracture healing. Methods Forty-eight rabbit models of femoral fracture were prepared and fixed with unilateral two-bar external fixator. They were randomly divided into four groups: continuing immobilization group, instant micromovement group, 1-week micromovement group and 2-week micromovement group. Postoperative radiographs were taken at 1, 2, 3 and 5 weeks to observe callus growth. The maximum load, deflection and rigidity of callus at fracture end were measured 5 weeks after operation. At 1, 2 and 3 weeks after operation, the histological morphology of callus was observed, and the expression and distribution of osteocalcin (oc) in callus were detected. Results At 5 weeks after operation, the X-ray scores of fracture line in 1-week micromovement group and 2-week micromovement group were 10.384±0.744 mm, 10.412±0.482 mm, significantly higher than those in continuing immobilization group (7.518±0.536). The anteroposterior diameter and the exterior and interior diameter of the external callus in 1-week micromovement group and 2-week micromovement group were 14.3±3.2 mm, 14.0±2.8 mm and 14.6±2.1 mm, 15.2±3.1 mm, which were smaller than those in the continuing immobilization group 15.3±2.3 mm and 16.7±1.9 mm, but there was no significant difference. The bone mineral density value and proportion rate in the fracture site were 0.446±0.020 g/cm2, 0.416±0.021 g/cm2 and 1.171%±0.056%, 1.143%±0.040% in 1-week micromovement group and 2-week micromovement group, which were significantly higher than those in continuing immobilization group which were 0.376±0.022 g/cm2 and 0.912%±0.051%. The maximum load of callus in 1-week micromovement group and 2-week micromovement group was 415.6±27.2 N, 400.3±28.5 N, which was significantly higher than that in continuing immobilization group 329.2±18.4 N and instant micromovement group 272.8±22.7 N. There was no difference of the deflection of callus between groups. The rigidity of callus in 1-week micromovement group was 590.4±24.2 N/mm, which was significantly higher than that in other groups; the rigidity of callus in the 2-week micromovement group was 540.6±22.8 N/mm, which was significantly higher than those in the instant micromovement group and the continuing immobilization group (152.4±21.7 N/mm, 174.8±20.6 N/mm). Conclusion Micromovement begins from one or two weeks can significantly raise external callus formation and vagueness level of fracture line, accelerating bridging callus formation, and can significantly raise bone mineral density and rigidity of callus. It also accelerates the maturity, hypertrophy and mineralization of chondrocyte, resulting in the stimulation of the fracture healing through endochondral ossification; it seemingly can improve the amount and density of osteoclasts in callus to stimulate the maturity and mineralization of chondrocyte. The strengthening coupling of osteoblasts and osteoclasts can promote the transformation from soft callus to hard callus and the remolding of hard callus. Key words: Fractures, bone; Fracture healing; External fixators

Comparison of the efficacy of non-operative and surgical treatment of distal clavicle fractures in older children

Objective To compare the clinical efficacy of non-operative and surgical treatment of distal clavicle fractures in older children. Methods From January 2011 to March 2018, 41 older children with distal clavicle fractures were treated. Among them, 24 cases underwent non-operative treatment, shoulder abduction brace fixation for 4 to 6 weeks (non-operation group); while 17 cases underwent surgical treatment, open reduction and Kirschner wire internal fixation of the fractures on the affected side, and shoulder abduction brace fixation for 4 to 6 weeks (operation group). The fracture healing time, Constant-Murley shoulder function score and complication rate of the two groups were compared and analyzed. Results All the 41 cases were follow-up for 3 to 17 months, with an average of 8.4 months. There was no significant difference in gender, age and fracture side between the two groups, so two groups were comparable. All the incisions in the operation group achieved primary healing. X-ray examination showed that there was continuous callus formation at the fracture end and Kirschner wire was removed. The fracture healing time was (44.3±6.7) days. No early complications such as infection occurred. In the non-operation group, X-ray examination showed bone healing at the fracture end. The healing time was (41.7±7.6) days. At the last follow-up, there were no complications such as malunion of fracture, premature epiphyseal closure and obvious shoulder dysfunction in both groups. Constant-Murley shoulder joint function score: non-operative group was (97.7±4.7) points, while operation group was (96.5±3.7) points. There was no significant difference between the two groups (P=0.32). Conclusion Non-operative treatment of the distal clavicle fractures in older children has the same effect as surgical treatment. However, non-operative treatment has the advantages of less trauma, low cost and no need of secondary surgical removal of internal fixation. Key words: Clavicle; Fractures,bone; Child

The benefits of foot calluses.

The benefits of foot calluses.

Somatic embryogenesis and regeneration of Kenyan wheat (Triticum aestivum L.) genotypes from mature embryo explants

Wheat (Triticum aestivum L.) improvement via genetic transformation depends on an efficient regeneration system for recovery of transgenic events. This study reports a somatic embryogenesis-based regeneration system for two Kenyan wheat genotypes (Eagle 10 and Njoro bread wheat II) from mature embryos. The study investigated the efficiency of mercuric chloride, commercial bleach, and chlorine gas in surface sterilizing explants prior to in vitro culture. Callus induction and somatic embryogenesis were done by culturing wheat mature embryos in Murashige and Skoog (MS) medium supplemented with 2, 4-dichlorophenoxyacetic acid (2,4-D) used either singly or in combination with 1-naphthaleneacetic acid (NAA), 4-chlorophenoxyacetic acid (4-CPA) or 6-benzylaminopurine (BAP). Embryo germination and plantlet recovery were done by culturing embryogenic callus in MS medium without plant growth regulators (PGRs). Chlorine gas was significantly (p<0.001) the most effective in surface sterilization and maintenance of explant viability. All 2, 4-D concentrations tested (1, 2, 4 and 8 mg/l) induced embryogenic callus. A significantly higher callus induction rate and callus fresh weight were obtained when 2,4-D was used in combination with either NAA or 4-CPA than when it was used singly. Combining 2,4-D with BAP led to a significantly lower callus induction frequency. Somatic embryo germination was achieved in MS medium without plant growth regulators. These findings have the potential to inform future efforts in the application of modern biotechnology for accelerated wheat cultivar improvement. Key words: Wheat, somatic embryogenesis, mature embryos.

Arthroscopic bone grafting with percutaneous fixation in treating scaphoid nonunion

Objective To analyze the clinical effects of arthroscopic autologous bone grafting and percutaneous fixation in treating scaphoid nonunion. Methods From May 2013 to August 2017, a total of 25 cases of patients including 20 males and 5 females with unilateral scaphoid fractures and nonunion were reviewed, with mean age of 35.80±2.41 years (18-65 years). The duration from injury to treatment was averaged 11.70±1.90 months (5-18 months). All of the cases sustained waist and proximal end fractures. X-ray and CT scan showed sclerosis and bone resorption without any callus at the fracture sites. However, there were no serious deformities and wrist arthritis. The patients suffered pain and weakness at the radial side of the wrist. The type of the fractures were Slade-Geissler's III-VI, including grade III 4 cases, grade IV 13 cases, grade V 7 cases and grade VI 1 case. The patients were treated with arthroscopic debridement of the sclerotic bone, autologous bone grafting, percutaneous screw (9 cases) or K-wires fixation (16 cases) and immobilization by plaster for 3 weeks after operation, followed by functional rehabilitation training. Bone union was assessed by serial plain radiographs and CT scan regularly. The functional effects were evaluated by comparing the modified Mayo wrist score with the visual analogue scale (VAS) for pain, range of motion (ROM) and the grip strength, which were measured before operation and at 18 months after operation. Results All cases were followed up. Bone union was achieved in all of 25 nonunion. The average radiological union duration was 10.24±2.10 weeks (6-20 weeks). The average VAS score decreased from 6.75±1.10 preoperatively to 1.33±0.21. The mean ROM of wrist was improved to 168.48°±12.41° (92.90% of that of the normal side), compared to that of 135.24°±17.47° preoperatively (79.80% of that of the normal side). The mean grip strength showed improvement from an average of 35.68±3.81 kg (80.46% of that of normal side) preoperatively to 48.75±4.42 kg (90.65% of that of normal side). The average modified Mayo wrist score improved from 61.52±6.32 preoperatively to 85.88±8.37. Conclusion Arthroscopic autologous bone grafting with percutaneous cannulated screw and K-wires fixation is an effective and minimally invasive treatment for scaphoid nonunion, which could protect the blood supply of the fracture sites, decrease the surgical complications, promote bone healing and lead to a faster recovery. Key words: Arthroscopy; Bone transplantation; Scaphoid bone; Fractures, ununited; Fracture fixation, internal

Minimally invasive treatment of double forearm fractures in children with elastic intramedullary nails

Objective To investigate the clinical efficacy and surgical techniques of elastic intramedullary nail in the treatment of double forearm fractures in children. Methods From March 2014 to July 2017, 12 children with double forearm fractures were treated with titanium elastic intramedullary nails. The elastic intramedullary nails were inserted from the distal radius and proximal ulna, and the affected limb was fixed with plaster over elbow and wrist for 4 to 6 weeks. When X-ray showed that the callus was well formed, the plaster was removed for functional exercise. The elastic intramedullary nails were removed after fracture healing. Results The closed reduction of radius and ulnar was performed in 7 and 8 cases respectively. The open reduction of radius and ulnar was performed in 5 and 4 cases respectively. The average length of incision at nail entry point was (1.17±0.17) cm. The average length of incision for open reduction was (1.54±0.29) cm. The operation time was (1.71±0.63) hours. The average amount of bleeding during operation was (12.92±7.53) ml. The postoperative follow-up period was 8 to 48 months, with an average of (23.58±11.42) months. The duration of bone healing was 3 to 8 weeks with an average of (4.92±1.38) weeks. The pronation angle of forearm was (83.75±6.44)°, and the supination angle was (85.83±5.15)°. The flexion and extension activities of elbow and wrist joints were similar to those of the healthy side. No complications such as nonunion, malunion, epiphyseal injury and needle tail irritation symptoms occurred in all the cases. Conclusion The application of titanium intramedullary nail is effective and minimally invasive in the treatment of double forearm fractures in children. Key words: Child; Forearm; Fractures,bone; Intramedullary nails

Hydroxyapatite/zirconia scaffold by three-dimensional printing compounded with vascular endothelial growth factor 165 calcium alginate microsphere slow-release system for repairing large bone defect in dogs

Objective To investigate the effect of hydroxyapatite/zirconia (HA/ZrO2) scaffold by three-dimensional printing compounded with vascular endothelial growth factor (VEGF) 165 calcium alginate microsphere slow-release system on repairing femoral shaft defects in dogs. Methods The HA/ZrO2 artificial prosthesis was prepared by three-dimensional printing, and the co-culture system of slow-release system of composite VEGF 165 calcium alginate microspheres was constructed. Sixteen beagle dogs were divided into four groups according to the extent of femoral shaft interception, with four dogs in each group. Group A: no biomaterials were implanted into the middle femur of dogs after 15 mm of femur interception as blank control group; Group B: HA/ZrO2 scaffolds composite with VEGF165 calcium alginate microspheres were implanted into the middle femur of dogs after 15 mm of femur interception; Group C: the same method as Group B was adopted after 25 mm of femur interception; Group D: the same method as Group B was adopted after 35 mm of femur interception. General examination and X-ray imaging observation were taken after operation. The ability of new HA/ZrO2 gradient biocomposites to repair bone defects was evaluated by micro CT scanning, biomechanical testing, ink staining and toluidine blue staining 12 weeks after operation. Results The drug loading capacity of calcium alginate microspheres reached (23.6±2.9)ng/mg, and the entrapment efficiency reached (62.4±3.6)%, showing a slow rate of release. Gross examination showed surgical incision was healed in all four groups. Postoperative X-ray imaging of experimental animals showed that nonunion was formed in Group A over time; in Group B, the artificial prosthesis was gradually filled with new bone and the boundary was blurred; in Group C, the early reaction was slower than that in Group A, and the callus passed continuously 12 weeks after operation; in Group D, new bone formation was slow, only surrounding the broken end. At 12 weeks after operation, the neonatal bone mass was (238.6±19.1)mm3 in Group B, (223.3±13.4)mm3 in Group C, and (110.8±6.5)mm3 in Group D. There were significant differences among the three groups (P 0.05). The results limit compression test at 12 weeks after operation showed no significant differences among Groups B [(49.7±2.3)MPa], C [(49.81±2.4)MPa] and D [(46.9±3.6)MPa] (P>0.05). At 12 weeks after operation, the histological sections showed that the blood vessels in Groups B and C were thickened, with obvious branches, and the surrounding new bone increased. During the period, the blood vessels were filled with vascular network. There were no obvious differences in the number and shape of blood vessels between groups. However, Group B had more new bones and blood vessel networks. New bone and small vascular networks were seen in Group D. Conclusion The hydroxyapatite/zirconia scaffold by three-dimensional printing compounded with vascular endothelial growth factor 165 calcium alginate microsphere slow-release system can repair dogs' femoral bone defect within 35 mm. Key words: Tissue engineering; Vascular endothelial growth factors; Three-dimensional printing technology

Embryogenesis in Valerian (Valeriana officinalis L.) using leaf segments

Abstract: A simple procedure for direct embryogenesis without an intervening callus production was developed in valerian (Valeriana officinalis L.) using the leaf segments. Direct somatic embryogenesis were induced using by half-strength MS medium supplemented with 2,4-D (0.5 mg·l -1 ), glutamic acid (100 mg·l -1 ), 4% sucrose and 8 g·l -1 agar. Germinated embryo to form plantlets enhanced on MS medium supplemented with NAA (0.1 mg·l -1 ) and Kin (2 mg·l -1 ). Regenerated plants with well-developed root and shoot systems were successfully (72%) transferred to greenhouse.

Micropropagation of two near threatened orchid. Part 2: Phalaenopsis amabilis Blume var. Grandiflora

Phalaenopsis is one of the most popular orchids in the world, through the development of many artificial hybrids. In this research, a reliable and efficient protocol is presented for in vitro proliferation of Phalaenopsis amabilis Blume cv. Grandiflora. Protocorm-like bodies (PLBs) were cultured on Murashige and Skoog (MS) medium containing different concentrations of kinetin (Kn; 0.00, 0.50, 1.00, 2.00 and 3.00 mg l-1) and indole-3-butyric acid (IBA; 0.00, 0.10, 0.20, 0.50 and 1.00 mg l-1), either individually or in combination and activated charcoal (AC; 0.00, 0.50 and 1.00 g l-1). A combination of 0.20 mg l-1 IBA and 2.00 mg l-1 Kn on medium containing 1.00 g l-1 AC was found to be suitable for maximum leaf number (6.16±0.503 per explant). The highest rooting frequency with 7.13±0.153 roots per explant was achieved on medium enriched with 0.50 mg l-1 IBA and 0.50 mg l-1 Kn on medium containing 1.00 g l-1 AC. The largest number of callus (9.10±0.611) was induced on explants cultured in medium containing 0.20 mg l-1 IBA and 0.50 mg l-1 Kn on medium without AC. The plantlets were successfully acclimatized in the greenhouse with a survival rate of 95% exhibiting normal developmental patterns.

Plant regeneration by organogenesis from bulbous explants in Fritillaria imperialis L., a wild rare ornamental species at the risk of extinction

Fritillaria imperialis L. (Liliaceae) is a rare and endangered ornamental plant grown in mountain regions and Zagros altitudes, Ilam province, Iran. This species is in danger of extinction due to invasive collection. Plant regeneration was done by organogenesis from bulb scales as explants cultured on Murashige and Skoog (MS) media fortified with different concentrations of kinetin (KIN, 0.00, 0.50, 1.00 and 2.00 mg l-1) and α_naphthaleneacetic acid (NAA, 0.00, 0.50, 1.00 and 2.00 mg l-1), either individually or in combination. The largest number of leaf (3.80), root (5.86) and callus (8.16) per explant was regenerated on the medium containing 0.50 mg l-1 KIN and 1.00 mg l-1 NAA. Maximum viability percentage (96.66%) was obtained in medium supplemented with 1.00 mg l-1 KIN. In vitro regenerated plantlets were cultivated in plastic pots containing peat moss and perlite (1:1). The plantlets were successfully acclimatized in an adaptation greenhouse with a survival rate of 95% exhibiting normal developmental patterns.