Wednesday, 22 February 2017
Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls
Research (Published online: 23-02-2017)
17. Identification of single nucleotide polymorphism in protein phosphatase 1 regulatory subunit 11 gene in Murrah bulls - Varsha Jain, Brijesh Patel, Farhat Paul Umar, H. M. Ajithakumar, Suraj K. Gurjar, I. D. Gupta and Archana Verma
Veterinary World, 10(2): 244-248
Aim: This study was conducted with the objective to identify single nucleotide polymorphism (SNP) in protein phosphatase 1 regulatory subunit 11 (PPP1R11) gene in Murrah bulls.
Materials and Methods: Genomic DNA was isolated by phenol–chloroform extraction method from the frozen semen samples of 65 Murrah bulls maintained at Artificial Breeding Research Centre, ICAR-National Dairy Research Institute, Karnal. The quality and concentration of DNA was checked by spectrophotometer reading and agarose gel electrophoresis. The target region of PPP1R11 gene was amplified using four sets of primer designed based on Bos taurus reference sequence. The amplified products were sequenced and aligned using Clustal Omega for identification of SNPs. Animals were genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using EcoNI restriction enzyme.
Results: The sequences in the NCBI accession number NW_005785016.1 for Bubalus bubalis were compared and aligned with the edited sequences of Murrah bulls with Clustal Omega software. A total of 10 SNPs were found, out of which 1 at 5’UTR, 3 at intron 1, and 6 at intron 2 region. PCR-RFLP using restriction enzyme EcoNI revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study.
Conclusion: A total of 10 SNPs were found. PCR-RFLP revealed only AA genotype indicating monomorphism in PPP1R11 gene of all Murrah animals included in the study, due to which association analysis with conception rate was not feasible.
Keywords: Murrah bulls, protein phosphatase 1 regulatory subunit 11, polymerase chain reaction, restriction fragment length polymorphism.
Tuesday, 21 February 2017
Outbreak of foot and mouth disease and peste des petits ruminants in sheep flock imported for immediate slaughter in Riyadh
Research (Published online: 22-02-2017)
16. Outbreak of foot and mouth disease and peste des petits ruminants in sheep flock imported for immediate slaughter in Riyadh - M. A. Mahmoud and S. A. Galbat
Veterinary World, 10(2): 238-243
Aim: To detect and identify the causative agent or agents of the following clinical symptoms which were fever, lack of appetite, salivation, vesiculation, erosions of the buccal mucosa, nose, and feet. The signs vary from mild to severe. The mortality rate of the disease is high. The morbidity rate reaches up to 100%. Sheep also show bloody diarrhea and rapid respiration. Sheep flock resident in El-Kharje Governorate.
Materials and Methods: A total of 50 serum samples and 50 buffy coat samples were collected from Marino sheep flock suffered from high mortalities, fever, lameness, diarrhea, stomatitis, and respiratory distress. PrioCHECK® foot and mouth disease virus (FMDV) nonstructural (NS) (marketable enzyme-linked immunosorbent assay [ELISA] kit) was used for revealing of the NS antibodies and liquid phase blocking enzyme immunoassay (LPBE) for identifying the FMD serotype and examined by competitive ELISA (cELISA) for detection of peste des petits ruminants (PPR) antibodies. The buffy coat samples were examined by immunocapture ELISA (Ic ELISA) for detection of PPR antigen.
Results: Using PrioCHECK® FMDV NS: Commercial ELISA kit: 38/50 (76%) of the serum samples were positive for the presence of FMD NS viral proteins. In addition, using LPBE the positive samples were identified as FMD serotype O. Examination of the serum sample by cELISA for detection of PPR antibodies gave positive results in 32/50 (64%). While the Ic ELISA identified 32 (64%) positive reactors for PPR antigen.
Conclusion: This study reflected high susceptibility of the imported sheep flocks to the infection with FMD and PPR viruses, which are endemic in the Kingdom of Saudi Arabia (KSA). Hence, the imported flocks that prepared for slaughter must be vaccinated with the used vaccine in KSA in the quarantine for the control of FMD especially when importation occurs from counters that are free from these diseases.
Keywords: foot and mouth disease, peste des petits ruminants, Marino sheep, virus, Kingdom of Saudi Arabia.
Monday, 20 February 2017
Research (Published online: 21-02-2017)
15. Potential antibacterial activity of some Saudi Arabia honey - Ahmed G. Hegazi, Faiz M. Al Guthami, Ahmed F. M. Al Gethami, Fyrouz M. Abd Allah, Ashraf A. Saleh and Ehab A. Fouad
Veterinary World, 10(2): 233-237
Aim: The aim of this study was to investigate the potential antibacterial activity of some Saudi Arabia honey against selected bacterial strains of medical importance.
Materials and Methods: A total of 10 Saudi Arabia honey used to evaluate their antimicrobial activity against some antibiotic-resistant pathogenic bacterial strains. The bacterial strains were Staphylococcus aureus, Streptococcus pyogenes, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa.
Results: The antibacterial activity of Saudi honey against five bacterial strains showed different levels of inhibition according to the type of honey. The overall results showed that the potential activity was differing according to the pathogen and honey type.
Conclusion: It could be concluded that the Saudi honey inhibit the growth of bacterial strains and that honey can be used as complementary antimicrobial agent against selected pathogenic bacteria.
Keywords: antibiotic-resistant, potential antibacterial activity, Saudi Arabia honey.
Sunday, 19 February 2017
Research (Published online: 19-02-2017)
14. Effect of various levels of date palm kernel on growth performance of broilers - Muhammad Hamza Tareen, Rani Wagan, Farman Ali Siyal, Daryoush Babazadeh, Zohaib Ahmed Bhutto, Muhammad Asif Arain and Muhammad Saeed
Veterinary World, 10(2): 227-232
Aim: The aim of this study was the assessment of various levels of date palm kernel (DPK) on the growth performance of broilers.
Materials and Methods: A 250-day-old broiler chicks were randomly selected and categorized into five groups (50 chicks/group) contained A (control), B, C, D and E fed with 0%, 1%, 2%, 3% and 4% levels of DPK in balanced ration, respectively, for 6 weeks. Feed and water intake were recorded daily in the morning and evening. The data for feed intake, water intake, live body weight, and feed conversion ratio (FCR) were recorded from all birds regularly. The carcass weight and percentage obtained via six slaughtered birds were randomly selected from each group. Finally, economic aspects of the rations evaluated.
Results: The most feed intakes of broilers were recorded in Group A (3915.1 g) that was significantly higher than Groups D and E. The highest water intake was in Group E (9067.78 ml) that was significantly higher than Group A and control group. Live body weight was highest in Group E (979.85 g) than Groups B, C, and control group. The best growth weights were determined significantly in Groups D (1921.96 g) and E (1935.95 g). The lowest FCRs were indicated significantly in Groups D (1.97 g/g) and E (1.92 g/g) than Groups B and A. The highest carcass weights were recorded in Groups D (1214.01 g) and E (1230.88 g) that were significantly more than other groups. Dressing percentages in Groups D (61.76%) and E (62.17%) were higher than other groups (p<0.05). The net profits (Rs.) in Groups A, B, C, D and E were indicated 27.01, 32.77, 36.78, 43.47 and 44.51 per broiler, respectively.
Conclusion: It was concluded that the high levels of DPK (3-4%) significantly decreased broiler feed intake and increased water intake, live body weight, growth weight, carcass weight, dressing percentage, net profit per bird and also had positive effects on growth of broilers.
Keywords: broiler, date palm kernel, growth performance.
Attribution of antibacterial and antioxidant activity of Cassia tora extract toward its growth promoting effect in broiler birds
Research (Published online: 19-02-2017)
13. Attribution of antibacterial and antioxidant activity of Cassia tora extract toward its growth promoting effect in broiler birds - Jyoti Sahu, K. M. Koley and B. D. Sahu
Veterinary World, 10(2): 221-226
Aim: The study was conducted to evaluate the attribution of antibacterial and antioxidant activity of methanolic extract of Cassia tora toward its growth promoting effect in broiler birds.
Materials and Methods: A limit test was conducted for C. tora extract in Wistar albino rats. Phytochemical screening of methanolic extract of leaves of C. tora was carried out. In-vitro antibacterial activity was measured by disc diffusion method. 1-day-old Ven Cobb broiler birds (n=90) were randomly allocated into three groups consisting of three replicates with 10 birds in each group. The birds of group T1 (Control) received basal diet, whereas birds of group T2 (Standard) received an antibiotic (Lincomycin at 0.05% in feed). The birds of group T3 (Test) received Cassia tora extract (CSE) at 0.4 g/L in drinking water in addition to basal diet. The treatment was given to birds of all the groups for 6 weeks. Antioxidant activity of C. tora was determined in blood of broiler birds. Cumulative body weight gain, feed intake, feed conversion ratio (FCR), dressing percent, and organ weight factor were evaluated to determine growth performance in broiler birds.
Results: Phytochemicals in C. tora were screened. Sensitivity to Escherichia coli and resistant to Staphylococcus aureus and Pseudomonas aeruginosa was observed in in-vitro antibacterial activity test. At the end of 6th week, antioxidant activity reflected significantly (p≤0.05) lower level of erythrocyte malondialdehyde and higher levels of reduced glutathione (GSH) and GSH peroxidase in broiler birds of group T2 and T3 as compared to broiler of group T1. Mean cumulative body weight gain of birds of T2 and T3 were significantly (p≤0.05) higher as compared to T1. Mean FCR of birds of group T3 decreased significantly than group T1.
Conclusion: Supplementation of C. tora leaves extract at 0.4 g/L in drinking water improved growth performance in broiler birds due to its antimicrobial and antioxidant activity. Therefore, it could be used as an alternative to antibiotic growth promoter in poultry ration.
Keywords: antibacterial activity, antioxidant activity, broiler birds, Cassia tora, growth promoter.
Friday, 17 February 2017
Biochemical components of seminal plasma and their correlation to the fresh seminal characteristics in Marwari stallions and Poitou jacks
Research (Published online: 18-02-2017)
12. Biochemical components of seminal plasma and their correlation to the fresh seminal characteristics in Marwari stallions and Poitou jacks - Thirumala Rao Talluri, Gorakh Mal and Sanjay Kumar Ravi
Veterinary World, 10(2): 214-220
Aim: To investigate various biochemical components of seminal plasma in Marwari stallions and Poitou Jacks and to find out their correlation with that of the seminal characteristics.
Materials and Methods: In this study, semen was collected from six Marwari stallions and six Poitou jacks aged from 4 to 6 years and with known fertility status. The semen collection from the stallions were collected during the breeding season, i.e., between the months of April and June. From the collected semen ejaculates, we estimated the values of some biochemical components, viz., total protein content, total lipid content, and enzymes such as glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), alkaline phosphatase (ALP), acid phosphatase (ACP), and lactate dehydrogenase (LDH) as well as concentrations of glucose, cholesterol, total calcium (Ca), and phosphorus (P) and correlations among different seminal parameters were statistically examined using the Pearson correlation coefficient.
Results: In this study, we found positive correlations between semen volume as well as sperm concentration and GOT, GPT, ALP and ACP for both the group stallions. Significant correlation between motility and glucose, GOT and GPT could be an indication for their role metabolism and protection against free radicals to the spermatozoa.
Conclusion: Based on the results, it is concluded that there is a positive correlation between some biochemical values such as glucose, Ca, ALP, and LDH and seminal parameters which play a key role in capacitation and onward movement of the spermatozoa.
Keywords: alkaline phosphatase, biochemical components, lactate-dehydrogenase, Marwari, Poitou, stallion, seminal plasma.
Research (Published online: 17-02-2017)
11. A combinatorial model for effective estrus detection in Murrah buffalo - Ramu Muthu Selvam and Govindaraju Archunan
Veterinary World, 10(2): 209-213
Background: Buffaloes are silent heat animals and lacunae in their estrus detection results a substantial economic loss in developing countries. Many advanced tools to aid heat detection have been developed but are neither affordable nor easily interpretable by marginal farmers.
Aim: The present investigation was made to develop a cost-effective estrus detection model by combining several known estrus predicting parameters.
Materials and Methods: Various signs of estrus were classified under major parameters such as visual, cow behavioral, bull behavioral, biochemical, and gyneco-clinical. Expression of those parameters was observed in buffaloes, and the percentage of positive estrus detection was calculated for each combination of estrus prediction parameters.
Results: The present result concludes that the model comprises of five parameters group with several signals with twentysix different combinations. It was observed that the expression of individual combinations and their corresponding estrus detection efficiency varies significantly, i.e., detection efficiency rises as the number of combination increases.
Conclusion: Combination of three parameters would provide an estrus detection efficiency >70% and suggested for an easy estrus detection. This would be a cost-effective model for farmers and benefits in enhancing buffalo population/reproduction.
Keywords: behavior, estrus detection, fern pattern, Flehmen, mounting, visual.
Thursday, 16 February 2017
Toxoplasma gondii infection in meat animals from Africa: Systematic review and meta-analysis of sero-epidemiological studies
Research (Published online: 16-02-2017)
10. Toxoplasma gondii infection in meat animals from Africa: Systematic review and meta-analysis of sero-epidemiological studies -Aretas Babatoundé Nounnagnon Tonouhewa, Yao Akpo, Philippe Sessou, Camus Adoligbe, Eric Yessinou, Yaovi Gildas Hounmanou, Marc Napoléon Assogba, Issaka Youssao and Souaïbou Farougou
Veterinary World, 10(2): 194-208
Aim: Toxoplasma gondii is an ubiquitous apicomplexan parasite which causes toxoplasmosis in humans and animals. Felids especially cats are definitive hosts and almost all warm-blooded mammals, including livestock and human can serve as intermediate hosts. Food animals can be reservoirs for T. gondii and act as one of the sources for parasite transmission to humans. The objective of this study is to collect serological data on the prevalence of anti-T. gondii antibody, and risk factors for certain food animals from Africa to provide a quantitative estimate of T. gondii infection among these species from different African countries.
Materials and Methods: Four databases were used to search seroepidemiological data on the prevalence of anti-T. gondii antibody in food animals between 1969 and 2016 from African countries. The search focused on data obtained by serologic test in food animals and meta-analyses were performed per species.
Results: A total of 30,742 individual samples from 24 countries, described in 68 articles were studied. The overall estimated prevalence for toxoplasmosis in chicken, camel, cattle, sheep, goat, pig were 37.4% (29.2-46.0%), 36% (18-56%), 12% (8-17%), 26.1% (17.0-37.0%), 22.9% (12.3-36.0%), and 26.0% (20-32.0%), respectively. Moreover, major risk factor of infection was age, farming system, and farm location.
Conclusions: A significant variation in the seroepidemiological data was observed within each species and country. The results can aid in an updated epidemiological analysis but also can be used as an important input in quantitative microbial risk assessment models. Further studies are required for a better and continual evaluation of the occurrence of this zoonotic infection.
Keywords: animal health, meta-analysis, Toxoplasmosis, zoonosis.
Monday, 13 February 2017
Preparation of mucosal nanoparticles and polymer-based inactivated vaccine for Newcastle disease and H9N2 AI viruses
Research (Published online: 14-02-2017)
9. Preparation of mucosal nanoparticles and polymer-based inactivated vaccine for Newcastle disease and H9N2 AI viruses - Heba M. El Naggar, Mohamed Sayed Madkour and Hussein Ali Hussein
Veterinary World, 10(2): 187-193
Aim: To develop a mucosal inactivated vaccines for Newcastle disease (ND) and H9N2 viruses to protect against these viruses at sites of infections through mucosal immunity.
Materials and Methods: In this study, we prepared two new formulations for mucosal bivalent inactivated vaccine formulations for Newcastle and Avian Influenza (H9N2) based on the use of nanoparticles and polymer adjuvants. The prepared vaccines were delivered via intranasal and spray routes of administration in specific pathogen-free chickens. Cell-mediated and humoral immune response was measured as well as challenge trial was carried out. In addition, ISA71 water in oil was also evaluated.
Results: Our results showed that the use of spray route as vaccination delivery method of polymer and nanoparticles MontanideTM adjuvants revealed that it enhanced the cell mediated immune response as indicated by phagocytic activity, gamma interferon and interleukin 6 responses and induced protection against challenge with Newcastle and Avian Influenza (H9N2) viruses.
Conclusion: The results of this study demonstrate the potentiality of polymer compared to nanoparticles adjuvantes when used via spray route. Mass application of such vaccines will add value to improve the vaccination strategies against ND virus and Avian influenza viruses.
Keywords: adjuvant, H9N2, mucosal, nanoparticles, Newcastle, polymer.
Profile and artificial insemination practices of technicians and the artificial insemination success rates in Leyte, Samar, and Biliran, Philippines (2011-2015)
Research (Published online: 13-02-2017)
8. Profile and artificial insemination practices of technicians and the artificial insemination success rates in Leyte, Samar, and Biliran, Philippines (2011-2015) - Adrian P. Ybañez, Rochelle Haidee D. Ybañez, Maxine O. Caindec, Louie V. Mani, Julius V. Abela, Edgar S. Nuñez, Johnson T. Royo Jr and Ivy Fe M. Lopez
Veterinary World, 10(2): 181-186
Background: Artificial insemination (AI) is a reproductive biotechnology that may be influenced by several factors, including the profile of the technicians and the practices used. Assessing technician’s profile and their AI practices can be significant in improving AI success rate.
Aim: This study aimed to know the profile and current practices used by AI technicians (AITs), to determine the success rates of AI in water buffaloes in Leyte, Samar, and Biliran from 2011 to 2015, and to evaluate the possible association between the parameters investigated.
Materials and Methods: A total of 50 AITs from Leyte, Samar and Biliran, Philippines, were interviewed using a fixed questionnaire about their profile and employed AI practices, and 20,455 AI-related records of the Philippine Carabao Center (PCC) at Visayas State University (VSU), Baybay City, Leyte, were screened and analyzed. AI success rates were determined by retrospective analysis of the gathered data. Statistical analysis was performed between the technician profile and practices and the AI success rates.
Results: Results revealed that most of the technicians were male, around 31-40 years old, married, college graduates, working under local government units, had other sources of income, and with 1-5 years of continuous AI practice averaging 51-100 inseminations per year. Most of them attended only one basic training seminar, which was conducted more than 3 years ago in PCC in VSU. AI success rates were recorded highest in 2011 and lowest in 2015. Statistical analyses showed that some technician profile parameters (civil status, average AI per year, and the training center) and several practices (checking of soft cervix, rectal palpation, thawing temperature method, straw cutting method, and semen deposition) might have an influence on the success of AI.
Conclusion: This study documents the first report on AIT’s profile and their employed AI practices and the AI success rates in Leyte, Samar, and Biliran, Philippines. Selected profile parameters and AI practices may influence AI success rates. AITs should perform more AI services and revisit the employed practices.
Keywords: artificial insemination, artificial insemination technician, Philippines, profile, water buffalo.
Sex-related differences in hemato-biochemical indices of adult Vanaraja chickens during summer and winter seasons
Research (Published online: 13-02-2017)
7. Sex-related differences in hemato-biochemical indices of adult Vanaraja chickens during summer and winter seasons - Kuldeep Kumar Panigrahy, Kumaresh Behera, Lal Mohan Mohapatra, Aditya Prasad Acharya, Kamdev Sethy, Sasmita Panda and Shailesh Kumar Gupta
Veterinary World, 10(2): 176-180
Aim: The objective of this study was to evaluate the changes in hemato-biochemical indices in male and female Vanaraja chickens under tropical environment during summer and winter season.
Materials and Methods: A total of 120 day-old sexed Vanaraja chicks were selected as experimental chickens and distributed equally in two groups having 60 female and 60 male chickens in each group, respectively. The experiment was continued for 8 weeks (56 days) and both male and female chickens were slaughtered by cervical dislocation method. All parameters were estimated at the end of the experiment in both seasons.
Results: Male had higher blood glucose, Ca and P level. Blood glucose level significantly (p<0.05) reduced in summer. Female had higher total protein, albumin, globulin, and albumin/globulin ratio. Alanine aminotransferase and aspartate aminotransferase enzyme concentration were significantly (p<0.05) higher in summer. Total erythrocyte count, total leukocyte count, hemoglobin (Hb), Hb/lymphocyte ratio, and packed cell volume were significantly (p<0.05) higher in males. Mean corpuscular volume and mean corpuscular Hb were significantly (p<0.05) higher in females.
Conclusion: Sex of chickens had a significant (p<0.05) effect on different parameters whereas season had nonsignificant (p>0.05) effect in most of the observed parameters. Hence, Vanaraja chickens are adaptable to local tropical climate and can be reared efficiently as backyard poultry.
Keywords: indices, season, tropical climate, Vanaraja chicken, welfare.
Saturday, 11 February 2017
Extraction of chitosan and its oligomers from shrimp shell waste, their characterization and antimicrobial effect
Research (Published online: 12-02-2017)
6. Extraction of chitosan and its oligomers from shrimp shell waste, their characterization and antimicrobial effect - Tarun Kumar Varun, Swaraj Senani, Natasha Jayapal, Jayaram Chikkerur, Sohini Roy, Vijay Bhasker Tekulapally, Mayank Gautam, and Narender Kumar
Veterinary World, 10(2): 170-175
Aim: The present study was performed to utilize the shrimp shell waste for chitin and chitosan production, characterization by Fourier transform infrared (FT-IR) technique and to evaluate the antimicrobial effects of chitosan oligomers produced by depolymerization of chitosan by nitrous acid.
Materials and Methods: Chitosan was extracted from the shrimp shell waste by the chemical method and characterized by FT-IR. Chitooligomers were produced by depolymerising chitosan using nitrous acid, and the chitooligomers were tested for antimicrobial effect against four gut pathogenic organisms, i.e., Enterobacter aerogen (National Collection of Dairy Culture [NCDC] 106), Enterococcus faecalis (NCDC 119), Escherichia coli (NCDC 134), and Staphylococcus aureus (NCDC 109) by well diffusion method using Muller-Hinton agar. A pure culture of pathogenic organisms was collected from NCDC, ICAR-National Dairy Research Institute, Karnal.
Results: Extracted chitosan characterized by FT-IR and chitooligomers demonstrated antimicrobial effect against four gut pathogenic organisms used in this study. Zone of inhibitions (mm) were observed in E. faecalis (13±0.20), E. coli (11.5±0.4), S. aureus (10.7±0.2), and E. aerogen (10.7±0.3). E. faecalis showed larger inhibition zone as compared to all other organisms and inhibitions zones of E. aerogen and S. aureus were comparable to each other.
Conclusion: Shrimp waste can be utilized for chitosan production, and the chitooligomers can be used as feed additive for gut health enhancement and have potential to replace antibiotics from the feed. Along with value addition pollutant load could be reduced by waste utilization.
Keywords: chitin, chitooligomers, chitosan, Fourier transform infrared, shrimp waste.
Wednesday, 8 February 2017
Evaluation of tissue-engineered bone constructs using rabbit fetal osteoblasts on acellular bovine cancellous bone matrix
Research (Published online: 08-02-2017)
5. Evaluation of tissue-engineered bone constructs using rabbit fetal osteoblasts on acellular bovine cancellous bone matrix - Rashmi, Rekha Pathak, Amarpal, H. P. Aithal, P. Kinjavdekar, A. M. Pawde, A. K. Tiwari, P. Sangeetha, P. Tamilmahan, and A. B. Manzoor
Veterinary World, 10(2): 163-169
Aim: The aim of this study was to generate composite bone graft and investigate the rabbit fetal osteoblasts adhesion, proliferation and penetration on acellular matrices of cancellous bone.
Materials and Methods: Acellular cancellous bone was prepared and developed as in the previous study with little modification. These matrices were decellularized by rapid freeze and thaw cycle. To remove the cell debris, they were then treated with hydrogen peroxide (3%) and ethanol to remove antigenic cellular and nuclear materials from the scaffold. Primary osteoblast cells were harvested from 20 to 22 days old rabbit fetal long and calvarial bone. These cells were cultured and characterized using a specific marker. The third passaged fetal osteoblast cells were then seeded on the scaffold and incubated for 14 days. The growth pattern of the cells was observed. Scanning electron microscope and hematoxylin and eosin staining were used to investigate cells proliferation.
Results: The cells were found to be growing well on the surface of the scaffold and were also present in good numbers with the matrix filopodial extensions upto inside of the core of the tissue.
Conclusion: Thus, a viable composite scaffold of bone could be developed which has a great potential in the field of bone tissue engineering.
Keywords: composite grafts, osteoblasts, tissue engineering.