The influence of M-CSF on fracture healing in a mouse model

Macrophage colony-stimulating factor 1 (M-CSF) is known to play a critical role during fracture repair e.g. by recruiting stem cells to the fracture site and impacting hard callus formation by stimulating osteoclastogenesis. The aim of this experiment was to study the impact of systemic M-CSF application and its effect on bony healing in a mouse model of femoral osteotomy. Doing so, we studied 61 wild type (wt) mice (18-week-old female C57BL/6) which were divided into three groups: (1) femoral osteotomy, (2) femoral osteotomy + stabilization with external fixator and (3) femoral osteotomy + stabilization with external fixator + systemic M-CSF application. Further, 12 op/op mice underwent femoral osteotomy and served as proof of concept. After being sacrificed at 28 days bony bridging was evaluated ex vivo with µCT, histological and biomechanical testing. Systemic M-CSF application impacted osteoclasts numbers, which were almost as low as found in op/op mice. Regarding callus size, the application of M-CSF in wt mice resulted in significantly larger calluses compared to wt mice without systemic M-CSF treatment. We further observed an anabolic effect of M-CSF application resulting in increased trabecular thickness compared to wt animals without additional M-CSF application. Systemic M-CSF application did not alter biomechanical properties in WT mice. The impact of M-CSF application in a mouse model of femoral osteotomy was oppositional to what we were expecting. While M-CSF application had a distinct anabolic effect on callus size as well as trabecular thickness, this on bottom line did not improve biomechanical properties. We hypothesize that in addition to the well-recognized negative effects of M-CSF on osteoclast numbers this seems to further downstream cause a lack of feedback on osteoblasts. Ultimately, continuous M-CSF application in the absence of co-stimulatory signals (e.g. RANKL) might overstimulate the hematopoietic linage in favor of tissue macrophages instead of osteoclasts.

Callus Induction and Virus Free Potato Mini-Tuber Production through Meristem Culture

The experiments were conducted with a view to establish a protocol for virus free mini-tubers production, in vitro, using potato meristem as explant, collected from potato sprouts of three popular potato varieties Diamant, Cardinal, Granola. In the present study four levels of GA3 (100, 200, 300 and 400 ppm) were used to assess the influence on sprouting abilities in three selected potato verities. The maximum sprouting efficiency was observed in 400ppm GA3 treatment within short period of time in Granola variety. The effect of different combination and concentration of IBA, 2, 4-D and BAP was used along with fresh MS media to inoculate meristems of potato sprouts. IBA concentration were 0.25mg/L, 1.5mg/L; 2, 4-D concentration were 0.125mg/L, 0.25mg/L, 0.37mg/L, 0.50mg/L, 1.0mg/L 2.0mg/L and BAP concentration were 0.5 mg/L, 1.0 mg/L, 1.5 mg/L and 2mg/L. The effect of combined hormones was also studied. The Diamant variety showed the maximum callus size (0.74cm) within a very short period of time (2 days) while treated at the concentration of 0.25 mg/L IBA, 0.25 mg/L 2, 4-D and 1.00 mg/L BAP. Granola meristem inoculated in hormonal treatment showed the best results regarding minimum days required to shoot initiation (5.34 days) followed by Diamant (6.68) days and Cardinal (7.64 days). The highest number of shoots/plantlet (4.33 shoots/plantlet) and the longest plantlet (9.32 cm) was found in Granola variety followed by Diamond (3.67 shoots/plantlet; 7.53cm) and Cardinal (4.0 shoots/plantlet; 7.62 cm). Maximum numbers of leaves were also found in Granola variety (15 leaves) in the treatment combination of 0.25 mg/L IBA, 0.125 mg/L 2,4-D and 0.5 mg/L BAP. Granola gave maximum performance in respect of maximum length of root (6.55 cm roots/plantlets) within a short time of root initiation (20 days), followed by Diamant (4 cm roots/plantlets) of root initiation (4.47 days) and Cardinal (4.53 cm roots/plantlets) of root initiation (26.39 days). Rooted plantlets were gradually acclimatized and successfully established in the field. However, in overall observation in this study, Granola showed better performance from meristem tissue culture and all plants were found normal and free from potato viruses that were tested through ELISA test.

Effect of plant growth regulators on plant regeneration of hyacinth bean (Lablab purpureus (L.) Sweet) through nodal explants

Nodal explants of three accessions namely BD-101, BD-122 and BD-8001 of hyacinth bean (Lablab purpureus (L.) Sweet) were cultured for four weeks on MS medium supplemented with different concentrations and combinations of NAA (0, 0.1 mgl-1) and BAP (0.5, 1.0 and 2.5 mgl-1) for plant regeneration. The highest shoot initiation was observed in 0.5 mgl-1 BAP while the lowest shoot initiation was found in 2.5 mgl-1 BAP with 0.1 mgl-1 NAA. Earlier shoot initiation was exhibited in 0.5 mgl-1 BAP with 0.1 mgl-1 NAA. The highest number of leaflets and higher shoot lengths were observed in MS medium. Comparatively higher number of shoots was found in BAP (0.5-2.5 mgl-1). The highest percentage of callus was initiated in medium supplemented with 1.0 mgl-1 BAP. Earlier callus initiation and larger callus size were found in combination of BAP (0.5-2.5 mgl-1) with 0.1 mgl-1 NAA. BD-122 cultured in MS medium was found superior for shoot regeneration through node culture. Four different concentrations of IBA (0.1-1.5 mgl-1) were used for rooting. The highest percentage (86.67 %) of rooting was found in MS medium containing 0.1 mgl-1 IBA at four weeks. Rooting frequency decreased with the increasing concentration of IBA. The accession BD-8001 had 99.60 % rooting in 0.5 mgl-1 IBA. The highest number of longest roots was exhibited in 0.1 mgl-1 IBA. The regeneration protocol developed from nodal explants has applicability in improvement of hyacinth bean. Bangladesh J. Agril. Res. 44(1): 27-42, March 2019

Callus Age and Size of Barley (Hordeum vulgare L.) Improves Regeneration Efficiency

In order to improve regeneration efficiency embryos derived from immature seeds of BARI barley-6 were taken for this study. In this case callus size, age and its fresh weight were considered. Embryos sizes were classified into four groups: 0.6-1.0 mm (A), 1.1-1.5 mm (B), 1.6-2.0 mm (C) and 2.1-2.5 mm (D), and cultured to semi-solid MS medium for callus induction. Five weeks old calli were transferred to MS medium that contained 1.0 mg/l BAP + 150 mg/l L-glutamine, for regeneration. Results indicated that 1.6-2.0 mm size of embryos produced the highest callusing (56.72%) and green plantlets (42.16%), while small sized (0.6-1.0 mm) showed very poor (2.75%) callusing and no regeneration occurred. Calli were divided into three age groups e.g. early (1-3 weeks), medium (4-6 weeks) and prolonged age (7-12 weeks) and cultured to regeneration medium. To observe the effect of calli weight on regeneration, they were grouped into four categories: I (50-100 mg), II (101-150 mg), III (151-200 mg) and IV (>201 mg). The highest regeneration and rooting were recorded when the age of callus was 4-6 weeks and its weight range was 151–200 mg (III). The lowest regeneration and rooting were found when 1-3 weeks old calli were used and its average weight was 50-100 mg (I).

Accelerated fracture healing with teriparatide

Satisfactory healing of the osteoporotic fracture is critically important to functional recovery, morbidity, and quality of life. Some therapies for osteoporosis may affect the processes associated with bone repair. For example, bisphosphonates in experimental models are associated with increased callus size and mineralization, reduced callus remodeling, and improved mechanical strength. Local and systemic bisphosphonate treatment may improve implant fixation. No negative impact on fracture healing has been observed, even after major surgery or when administered immediately after fracture. For the osteoanabolic agent teriparatide, case reports and a randomized trial have produced mixed results, but they are consistent with a positive impact of teriparatide on fracture healing. Some of the agents currently being developed for osteoporosis, notably sclerostin and DKK1 antibodies have shown a beneficial effect on fracture healing. At this point, therefore, there is no evidence that osteoporosis therapies are detrimental to fracture healing with some promising experimental evidence for positive effects on healing, notably for those agents whose actions are primarily anabolic.

Low-level mechanical stimulation is sufficient to improve tendon healing in rats

Treatment of tendon injuries often involves immobilization. However, immobilization might not prevent mild involuntary isometric muscle contraction. The effect of weak forces on tendon healing is therefore of clinical interest. Studies of tendon healing with various methods for load reduction in rat Achilles tendon models show a consistent reduction in tendon strength by at least half, compared with voluntary cage activity. Unloading was not complete in any of these models, and the healing tendon was therefore still exposed to mild mechanical stimulation. By reducing the forces acting on the tendon even further, we now studied the effects of this mild stimulation. Rat Achilles tendons were transected and allowed to heal spontaneously under four different loading conditions: 1) normal cage activity; 2) calf muscle paralysis induced by botulinum toxin A (Botox); 3) tail suspension; 4) Botox and tail suspension, combined, to eliminate even mild stimulation. Healing was evaluated by mechanical testing after 8 days. Botox alone and suspension alone both reduced tendon callus size (transverse area), thereby impairing its strength compared with normal cage activity. The combination of Botox and suspension did not further reduce tendon callus size but drastically impaired the material properties of the tendon callus compared with each treatment alone. The peak force was only a fifth of that in the normal cage activity group. The results indicate that also the mild loading that occurs with either Botox or suspension alone stimulates tendon healing. This stimulation appears to affect mainly tissue quality, whereas stronger stimulation also increases callus size.