Robert M. Leven

Locally delivered rhTGF-β2 enhances bone ingrowth and bone regeneration at local and remote sites of skeletal injury

The purposes of the present study were to determine if recombinant human transforming growth factor‐beta‐2 (rhTGF‐β2) enhances bone ingrowth into porous‐coated implants and bone regeneration in gaps between the implant and surrounding host bone. The implants were placed bilaterally for four weeks in the proximal humeri of skeletally mature, adult male dogs in the presence of a 3‐mm gap. In three treatment groups of animals, the test implant was treated with hydroxyapatite/tricalcium phosphate (HA/TCP) and rhTGF‐β2 in buffer at a dose per implant of 1.2 μg (n = 6), 12 μg (n = 7), or 120 μg (n = 7) and placed in the left humerus. In these same animals, an internal control implant treated only with HA/TCP and buffer was placed in the right humerus. In a non‐TGF‐β treated external control group of animals (n = 7), one implant was treated with HA/TCP while the contralateral implant was not treated with the ceramic. In vitro analyses showed that approximately 15% of the applied dose was released within 120 h with most of the release occurring in the first 24 h. The TGF‐μ treated implants had significantly more bone ingrowth than the controls with the greatest effect in the 12 μg/implant group (a 2.2‐fold increase over the paired internal control (P = 0.004) and a 4‐fold increase over the external control (P < 0.001)). The TGF‐β treated implants had significantly more bone formation in the gap than the controls with the greatest effect in the 12 and 120 μg groups (1.8‐fold increases over the paired internal controls (P = 0.003 and P = 0.012, respectively) and 2.8‐fold increases over the external controls (P < 0.001 and P = 0.001, respectively)). Compared to the external controls, the internal control implants tended to have more bone ingrowth (1.9‐fold increase, P = 0.066) and had significantly more bone formation in the gap (1.7‐fold increase, P = 0.008). Thus, application of rhTGF‐β2 to a porous‐coated implant‐stimulated local bone ingrowth and gap healing in a weakly dose‐dependent manner and stimulated bone regeneration in the 3‐mm gap surrounding the contralateral control implant, a site remote from the local treatment with the growth factor.

Immunomagnetic bead isolation of megakaryocytes from guinea-pig bone marrow : effect of recombinant interleukin-6 on size, ploidy and cytoplasmic fragmentation

Guinea‐pig bone marrow megakaryocytes were isolated using an antibody to platelet glycoprotein Ib and a second antibody conjugated to magnetic beads. The procedure yielded an average of 644 800 megakaryocytes from two guinea‐pigs with an average viability of 83%. All of the platelet glycoprotein Ib positive cells also expressed the platelet glycoprotein IIb–IIIa complex. The size and ploidy of megakaryocytes isolated by this technique were analysed in the presence of 10 ng/ml of interleukin‐6 (IL‐6). Without IL‐6 megakaryocyte size increased significantly after 24 h. but an even larger increase in size occurred in the presence of IL‐6. The modal ploidy class was 16N with an average of 19% 2N. 2·6% 4N, 16·4% 8N. 50·8% 16N and 11·1% 32N cells as determined by flow cytometry. Measurements made by microspectrophotometry were in close agreement. After 24 h incubation there was a significant rise in the percentage of 2N and 32N cells. The ploidy distribution after 24 h with IL‐6 was the same as the control. Megakaryocytes cultured in the absence of serum on collagen gels did not form pseudopods and fragment, as occurs with serum (Leven et al. 1987). Addition of IL‐6 to the serum‐free cultures caused megakaryocytes to form extensive proplatelet extensions. We conclude that large numbers of pure guinea‐pig bone marrow megakaryocytes can be isolated by immunomagnetic bead selection, including low ploidy immature megakaryocytes. Spontaneous maturation occurred as evidenced by the increase in megakaryocyte size and ploidy. IL‐6 altered megakaryocyte size and morphology but not ploidy, indicating that these different characteristics of megakaryocytes may be regulated separately.

Assessment of Axonal Growth into Collagen Nerve Guides Containing VEGF-Transfected Stem Cells in Matrigel

The early events associated with axonal growth into 10‐mm nerve gaps were studied histologically in the rat sciatic nerve model to determine if the outgrowth of blood vessels, Schwann cells, and axons could be enhanced. In the first two experimental groups, collagen nerve guides were filled with either saline or Matrigel. Marrow‐derived mesenchymal stem cells (MSCs) were added to Matrigel in two other groups, one of which contained cells transfected with VEGF (MSC/VEGF). After 21 days, the injury site was exposed, fixed, sectioned, and volume fractions of the conduit contents were determined by point counting. The bioresorbable collagen conduits appropriately guided the axons and vessels in a longitudinal direction. The volume fraction of axons was significantly greater in the group with saline when compared with all three groups with Matrigel. This measure had a significant positive correlation with actual counts of myelinated axons. The blood vessel volume fraction in the Matrigel group decreased compared with the saline group, but was restored in the MSC/VEGF group. All Matrigel groups had comparable cellularity and showed a distribution of residual Matrigel in acellular zones. The saline group, by contrast, sustained a network of delicate fibroblastic processes that compartmentalized the nerve and its natural matrix as it became infiltrated by axons as minifascicles. In conclusion, the reduction of axonal outgrowth in the Matrigel groups, when compared with the saline group, suggests that Matrigel may impede the early regenerative process even when enriched by the addition of MSCs or VEGF‐transfected cells. Anat Rec, 2009.

Ultrasound Enhances Recombinant Human BMP-2 Induced Ectopic Bone Formation in a Rat Model

Two methods to improve bone repair include the use of recombinant human bone morphogenetic protein-2 (rhBMP-2) and low-intensity pulsed ultrasound (LIPUS). The present study was designed to determine if LIPUS enhances the effect of rhBMP-2-induced bone formation in a well characterized ectopic implant model. Absorbable collagen sponges loaded with 0-, 1-, 2.5- or 5-microg doses of rhBMP-2 were implanted subcutaneously in 11-week-old, male Long Evans rats, followed by daily 20-min LIPUS or sham LIPUS treatment beginning 1 d after surgery. Explanted sponges were assessed for bone volume, mineral density and mineral content by microcomputed tomography (microCT). At two weeks, LIPUS had no effect on rhBMP-2-induced bone formation, but at four weeks, LIPUS increased bone volume in the 1-microg rhBMP-2-treated implants 117.7-fold (0.02 +/- 0.04 mm(3)vs. 2.07(S.E.M.) +/- 1.67 mm(3);p = 0.028), and 2.3-fold in the 5-microg dose implants (5.96 +/- 3.68 mm(3)vs. 13.52 +/- 6.81 mm(3);p = 0.077) compared with sham LIPUS. Bone mineral density was not affected by LIPUS treatment. Total mineral content followed the same pattern as bone volume. Histologic staining for mineralized tissue was consistent with the microCT observations. The present study is the first to demonstrate that LIPUS enhances bone formation induced by rhBMP-2.

Microstructural and strength evaluation of regenerate tissue during the consolidation period after vertical mandibular ramus distraction

Mandibular ramus height restoration by distraction osteogenesis (DO) is a key procedure in mandibular hypoplasia reconstruction. The objective of this study was to evaluate short-term skeletal changes in the regenerated bone after vertical mandibular ramus DO using a buried distraction device. Eight subadult beagle dogs underwent bilateral vertical mandibular ramus DO. After a 7-day latency period, distraction was performed at a rate of 0.5 mm twice a day for 12 days. Four dogs were killed at 1 month and four dogs at 2 months after the end of distraction. One intact beagle was included as an unoperated control. After sacrifice, micro computed tomography (μCT) and mechanical testing of distracted sites were used to measure bone volume (BV), total volume (TV), and mechanical peak load strength, respectively. The μCT images showed wide variation in the response, with some animals demonstrating considerable bone formation and reconstitution of the canal for the inferior alveolar nerve. Quantitatively, BV was no more than 67% and BV/TV was less than 25% of the intact control, and strength was approximately 33% of the intact control value. The 1 and 2 month values were similar. These results suggest that internal distractors can successfully reconstitute bone but that the regenerated tissue did not regain structural and mechanical characteristics of native bone within the 2 month study period.

Morphological and kinetic abnormalities of platelets in hypercholesterolemic rabbits

Hypercholesterolemia (HC = hypercholesterolemia or hypercholesterolemic) was produced in rabbits by feeding them diets supplemented with cholesterol and peanut oil. Platelet counts and volumes, white cell counts, reticulocyte counts, and hematocrits were determined at intervals for 8-12 weeks in blood from HC animals and controls on a normal rabbit diet. Microthrombocytosis was a consistent occurrence in the presence of HC, developing as early as 2 weeks into the diet. Microthrombocytosis was generally associated with normal platelet counts, but mild thrombocytosis occurred late in the diet at the time of the highest levels of serum cholesterol (greater than 1300 mg/dl). Platelets from HC rabbits were morphologically normal by transmission electron microscopy. Survivals of 51Cr-labeled platelets from HC and non-HC rabbits were measured in HC and non-HC recipients. The results identified an intrinsic defect in the ability of HC platelets to survive in the circulation. They also confirmed previous findings of an environmental defect in HC that causes shortened platelet survival.

α-Actinin arcs in megakaryocyte spreading☆

Abstract Cultured megakaryocytes, isolated from guinea pig bone marrow, were treated with buffer or adenosine diphosphate (ADP) (10 μM) on plain or coated glass surfaces. Control cells were rounded and non-adherent. The nucleotide induced the cells to spread to several times the initial diameter, and to become flattened and markedly adherent to the substratum. ‘Cytoskeletons’ were examined by transmission electron microscopy (TEM). Those from unspread cells contained only rare microfilaments and no filament bundles; those from spread cells contained large numbers of microfilaments in nets and many filament bundles, which were largely oriented circumferentially. Interference reflection microscopy demonstrated that the spread cells were attached to the substratum in arc-shaped regions, which corresponded to arcs containing alpha-actinin as seen by specific immunofluorescence of the same cells. However, other arcs of α-actinin staining did not correspond to arcs of tight attachment. We conclude that fibrous arcs, which appear to assemble as part of the spreading process, play a role in what are probably transient surface attachment sites. A survey of observations of spreading in other cell types suggests that similar arcs are more widespread than has been realized.

The behavioral and immunological effect of GM-1 ganglioside on nerve root regeneration following C5 nerve root avulsion in a rat model.

Preganglionic nerve root avulsion precludes sensory return, but motor regeneration is possible with sparing of motoneurons. The effect of GM-1 ganglioside treatment was studied with parallel evaluation of the autoimmune response. Rats (N=64) received injections of either GM-1 ganglioside or saline for 30 days following either C5 root avulsion or a hemilaminectomy control. The Bertelli grooming test assessed functional return. Before sacrifice at 5 months, serum was collected for enzyme-linked immunoabsorbent assay testing. Only 44% of the rats treated with ganglioside had a good functional outcome compared with 50% for controls. Although 17% of the rats developed anti GM-1 antibodies, there was no functional or histological evidence of neuropathy in any of the rats. We conclude that ganglioside treatment did not enhance recovery from peripheral nerve injury. Although an immune response was present in some rats, no overt signs of neuropathy were observed.

Tenascin-C is synthesised and secreted by megakaryocytes, whose adherence to intact tenascin is mediated by the integrin subunit α1

Bone marrow megakaryocytes reside on a complex extracellular matrix, produced by stromal cells, as well as by megakaryocytes themselves. Using immunofluorescence microscopy we have determined the distribution of tenascin-C in guinea-pig bone marrow, finding that it has a prominent fibrillar distribution in association with megakaryocytes. Western blot analysis of CHRF-288 cells, a human megakaryocytic cell line and isolated purified guinea-pig megakaryocytes, with polyclonal and monoclonal antibodies, demonstrate the presence of three tenascin isoforms (210, 220 and 250 kDa). Immunoprecipitation of radiolabelled isolated guinea-pig megakaryocytes cultured on a type I collagen gel, demonstrate that these three tenascin isoforms were present in mega-karyocytes, whereas only two isoforms were secreted into the media (210 and 220 kDa). Analysis of rat bone marrow by in situ hybridisation reveals that transcripts for tenascin-C are present in megakaryocytes. Integrin regulation of CHRF cell adhesion to intact tenascin was shown to be mediated solely by theβ1 integrin.

Lipid and membrane fluidity abnormalities in platelets and megakaryocytes of the hereditary macrothrombocytopenic Wistar Furth rat

Biochemical and functional abnormalities of megakaryocytes and platelets were studied in Wistar Furth (WF) rats which have genetically determined macrothrombocytopenia and megakaryocytopenia, and were compared with their counterparts in Sprague-Dawley (SD) rats. Both megakaryocytes and platelets synthesized phospholipids from [14C]acetate. WF and SD megakaryocytes incorporated 0.27 and 0.29 nmol acetate per 10(6) cells, respectively. Phosphatidylcholine (PC) accounted for 64% and 58% of the PL radioactive label in megakaryocytes of SD and WF rats, respectively, (P less than 0.05), while 69% of labeled activity was associated with PC of SD platelets compared to 60% found in PC of WF platelets (P less than 0.01). In WF platelets a significant increase in the levels of lysophosphatidylcholine (6.1% vs. 3.0%) was observed. WF platelets had substantially higher levels of esterified cholesterol, triglycerides, ceramides and a 3-fold increase in the total protein per platelet compared to SD platelets. The fatty acid composition of WF platelet PC showed quantitative abnormalities. Plasma lecithin-cholesterol acyl transferase activity and platelet function monitored by the uptake and release of [14C] serotonin showed nonsignificant variations between SD and WF rats. Compared with the control, platelet membrane fluidity, measured by fluorescence polarization using platelets labeled with 1,6-diphenyl-1,3,5-hexatriene, was significantly decreased in the WF rats.