Zbigniew Gugala

In vitro growth and activity of primary chondrocytes on a resorbable polylactide three-dimensional scaffold†

Sheep articular chondrocytes were cultured for 3, 6, and 9 weeks on a three-dimensional porous scaffold from poly(L/DL-lactide) 80/20%. Cell growth and activity was estimated from the amount of proteoglycans attached to the polylactide scaffold and the amounts of DNA and proteins measured in the cell lysate. Cell morphology was assessed from scanning electron microscopy. Histochemical staining of proteoglycans present in the sponge was used to visualize the chondrocyte ingrowth in the scaffold. The amounts of DNA, proteins, and proteoglycans increased with time of culturing. Chondrocytes on the polylactide scaffold maintained their round shape. The cell ingrowth into the sponge progressed with time of culturing and proceeded from the upper surface of the sponge toward its lower surface. At 9 weeks, the chondrocytes filled the whole scaffold and reached the opposite side of the sponge. The proteoglycans network was, however, more dense at the upper half of the scaffold.

Use of a Chimeric Adenovirus Vector Enhances BMP2 Production and Bone Formation

Recombinant adenoviral vectors have potential for the treatment of a variety of musculoskeletal defects and such gene therapy systems have been a recent research focus in orthopedic surgery. In studies reported here, two different adenovirus vectors have been compared for their ability to transduce human bone marrow mesenchymal stem cells (hBM-MSCs) and elicit bone formation in vivo. Vectors consisted either of standard adenovirus type 5 (Ad5) vector or a chimeric adenovirus type 5 vector that contains an adenovirus type 35 fiber (Ad5F35), which has been recently demonstrated to bestow a different cellular tropism, and a complete cDNA encoding human bone morphogenetic 2 (BMP2). Studies were also conducted to compare the transduction efficiency of these vectors using enhanced green fluorescent protein (GFP). hBM-MSCs transduced with Ad5F35 vectors had higher levels of transgene expression than those transduced with Ad5 vectors. The results also demonstrate that hBM-MSCs lack the coxsackie-adenovirus receptor (CAR), which is responsible for cellular adsorption of Ad5. Therefore, the data suggest that Ad5 virus adsorption to hBM-MSCs is inefficient. Ad5BMP2- or Ad5F35BMP2-transduced hBM-MSCs were also compared in an in vivo heterotopic bone formation assay. Mineralized bone was radiologically identified only in muscle that received the Ad5F35BMP2 transduced hBM-MSCs. In summary, Ad5F35BMP2 can efficiently transduce hBM-MSCs leading to enhanced bone formation in vivo.

Osteoinduction by ex vivo adenovirus-mediated BMP2 delivery is independent of cell type

The objective of the study was to analyze and compare the abilities of various human cell types with inherently dissimilar osteogenic potentials to induce heterotopic bone formation following ex vivo transduction with two distinct adenoviral vectors encoding bone morphogenetic protein type 2 (BMP2). The cells comprised primary human bone marrow mesenchymal stem cells (BM-MSCs), primary human skin fibroblasts (SFs), and a human diploid fetal lung cell line (MRC-5). The vectors included adenovirus type 5 or a chimeric adenovirus type 5 with the fiber gene of adenovirus type 35 (Ad5F35-BMP2), both demonstrating significantly different expression of BMP2 in vitro. The experimental groups consisted of the three human cell types transduced with each of the two adenoviral vectors. Using nonobese diabetic severe combined immunodeficiency (NOD/SCID) mice, the transduced cells were injected intramuscularly following ex vivo adenoviral transduction. The nature and extent of heterotopic bone formation were analyzed radiographically and histologically. At 14 days postinjection, abundant, highly mineralized bone was formed in mice injected with Ad5F35-BMP2-transduced cells irrespective of the cell type. There was no statistically significant difference in the amount of bone formed between BM-MSCs, SFs, and MRC-5 cells transduced with Ad5F35-BMP2, as assessed from bone surface area on biplanar plain radiography. Substantially lesser amounts or no bone could be detected in mice injected with cells transduced with Ad5-BMP2. Immunohistochemical analysis confirmed the presence of human cells in muscle as early as 2 days postdelivery; however, at 6–7 days after injection, the transduced cells could not be detected in surrounding muscle, or in the heterotopic bone, indicating the host origin of the newly formed bone. The results of the study demonstrate no significant difference in osteoinductive properties between BM-MSCs, SFs, and MRC-5 cells transduced ex vivo with the same type of adenovirus encoding BMP2. The level of BMP2 expression appears to be a crucial factor determining the extent of heterotopic bone formation and was significantly affected by the type of adenovirus used. In the cell types studied, Ad5F35-BMP2 was more efficacious than Ad5-BMP2 in providing adequate levels of BMP2 for efficient osteoinduction.

Adenovirus-mediated BMP2 expression in human bone marrow stromal cells.

Recombinant adenoviral vectors have been shown to be potential new tools for a variety of musculoskeletal defects. Much emphasis in the field of orthopedic research has been placed on developing systems for the production of bone. This study aims to determine the necessary conditions for sustained production of high levels of active bone morphogenetic protein 2 (BMP2) using a recombinant adenovirus type 5 (Ad5BMP2) capable of eliciting BMP2 synthesis upon infection and to evaluate the consequences for osteoprogenitor cells. The results indicate that high levels (144 ng/ml) of BMP2 can be produced in non‐osteoprogenitor cells (A549 cell line) by this method and the resultant protein appears to be three times more biologically active than the recombinant protein. Surprisingly, similar levels of BMP2 expression could not be achieved after transduction with Ad5BMP2 of either human bone marrow stromal cells or the mouse bone marrow stromal cell line W20‐17. However, human bone marrow stromal cells cultured with 1 μM dexamethasone for four days, or further stimulated to become osteoblast‐like cells with 50 μg/ml ascorbic acid, produced high levels of BMP2 upon Ad5BMP2 infection as compared to the undifferentiated cells. The increased production of BMP2 in adenovirus transduced cells following exposure to 1 μM dexamethasone was reduced if the cells were not given 50 μg/ml ascorbic acid. When bone marrow stromal cells were allowed to become confluent in culture prior to differentiation, BMP2 production in response to Ad5BMP2 infection was lost entirely. Furthermore, the increase in BMP2 synthesis seen during differentiation was greatly decreased when Ad5BMP2 was administered prior to dexamethasone treatment. In short, the efficiency of adenovirus mediated expression of BMP2 in bone marrow stromal cells appears to be dependent on the differentiation state of these cells. J. Cell. Biochem. 82: 11–21, 2001.

Healing of critical-size segmental bone defects in the sheep tibiae using bioresorbable polylactide membranes

Bioresorbable polylactide membranes were used to treat critical-size segmental defects in the sheep tibiae. Six different experimental groups (3 animals in each) were investigated. These included defects treated with polylactide membranes with or without perforations, single-or double-tube membrane designs, and were treated with or without cancellous bone grafting. Defect healing was assessed radiographically (plain radiography, CT) and histologically at 16 weeks post implantation. In all animals with bone defects treated with membranes without bone grafting, a non union developed and persisted until 16 weeks. Defect healing was only observed in the animals in which autogenous bone graft was used along with perforated membranes. The presence of perforations improved bone graft reconstitution as compared with historical controls (bone graft in combination with non perforated membranes). There was more bone formed in the defects covered with the double membranes (tube-in-tube) than in those covered with the single tube, despite higher graft volume used in the latter. In the defects covered with tube-in-tube membranes, new bone was formed preferentially within the space between the tubes resulting in a radiographically and histologically denser appearance. The thickness of new bone between the tubes had a thickness corresponding to that of the intact cortex. The study demonstrates that the tube-in-tube membrane is superior to the single membrane design with regard to the nature and extent of defect healing. The perforated polylactide membranes seem to enhance reconstitution of the bone graft within the defect. Further studies are required to better understand the mechanisms of bone graft reconstitution in the critical-size segmental bone defects and the role of perforated polylactide membranes in this process.

The Cylindrical Titanium Mesh Cage for Treatment of a Long Bone Segmental Defect : Description of a New Technique and Report of Two Cases

This report describes a new technique for treatment of a segmental defect in long bones that uses a cylindrical titanium mesh cage, in combination with cancellous bone allograft and demineralized bone matrix putty (Grafton), stabilized with a statically locked intramedullary nail. Two clinical cases of tibia defects treated with this technique are presented. At the one-year follow-up, radiographically both cases demonstrated excellent limb alignment, stability, and bony healing. Immediate full weight-bearing was initiated in each case, and early limb functional recovery was achieved. Preliminary data suggest that this technique may be a reasonable alternative to currently used methods for management of select long bone segmental defects.

Tibial intramedullary nail distal interlocking screw placement: comparison of the free-hand versus distally-based targeting device techniques

Intramedullary nailing is the standard treatment for closed and some open unstable diaphyseal tibia fractures. Fluoroscopy, while essential for proper nail placement can subject the surgical team and patient to substantial radiation. A new targeting system for tibia nail distal interlocking was developed by Orthofix to limit fluoroscopy. This prospective clinical study compares the Orthofix targeting system versus a free-hand technique for the tibial nail distal interlocking. Fifty eight consecutive patients with sixty tibial fractures amenable for nail fixation were randomly assigned into two equal groups: Group 1: Orthofix distally based distal targeting device and Group 2: a free-hand technique. In all the cases stabilization was achieved with a reamed statically locked tibial nail. Recorded data included accuracy of screw placement, duration of surgery prior to and during distal interlocking, and the fluoroscopy time prior to and during distal interlocking. Both groups revealed comparable fracture patterns. In all fractures the technical aspects of the surgical treatment were performed without complications. There was no statistically significant difference between the groups in the mean time of surgery prior to (62.02 vs. 61.01 min, P=0.92) and during distal interlocking (17.06 vs. 19.08 min, P=0.55), or in the total surgical time (81 vs. 85 min), respectively. Neither was there a statistically significant difference in the mean fluoroscopy time prior to distal interlocking (69 vs. 81 s, p=0.22) nor in the total fluoroscopy time (84 vs. 117 s). There was however, a statistically significant difference between the Orthofix and free-hand groups with regards to the mean fluoroscopy time during distal interlocking (15 vs. 36 s, P=0.01, respectively). This study demonstrates that the distally based distal targeting device by Orthofix for tibial nailing can significantly decrease the mean fluoroscopy time necessary to complete distal interlocking versus free-hand technique.

SEMAC-VAT and MSVAT-SPACE sequence strategies for metal artifact reduction in 1.5T magnetic resonance imaging.

Objectives:To evaluate the ability of four magnetic resonance imaging (MRI) techniques to correct for metallic artifacts. These techniques consisted of 3 2D techniques and one 3D technique. In 2D imaging the techniques View Angle Tilting (VAT), Slice Encoding for Metal Artifact Correction (SEMAC) and a technique that employed a combination of the first two (SEMAC-VAT) were evaluated. In 3D imaging the technique Multiple Slab acquisition with VAT based on a SPACE sequence was evaluated (MSVAT-SPACE). Materials and Methods:Agarose phantoms and tissue phantoms with two commonly used metal implants (stainless steel and titanium) as well as two volunteers with metal implants were imaged at 1.5T. All phantoms and volunteers were imaged using VAT, SEMAC, SEMAC-VAT and MSVAT-SPACE techniques, as well as 2D and 3D conventional imaging techniques. Each technique was optimized for different image contrast mechanisms. Artifact reduction was quantitatively assessed in the agarose phantoms by volumetric measurement. Image quality was qualitatively assessed by blinded reads employing two readers. Each reader independently viewed the tissue phantom images and in vivo human images. Statistical analysis was performed using a Friedman test, Wilcoxon test and weighted Cohens kappa test. Results:T1-weighted, T2-weighted, PD-weighted and STIR image contrasts were successfully implemented with the evaluated artifact reduction sequences in both the phantom experiments and in vivo images. For all evaluated image contrasts and both metal implants, a reduction in the volume of metal artifacts was seen when compared with 2D conventional acquisitions. The 2D metal artifact volumes on average were reduced by 49% ± 16%, 56% ± 15% and 63% ± 15% for VAT, SEMAC and SEMAC-VAT acquisitions respectively. When Friedman and Wilcoxon tests were applied the difference in metal artifact volume was found to be statistically significant when VAT, SEMAC and SEMAC-VAT were compared with the 2D conventional techniques. In 3D imaging on average MSVAT-SPACE reduced metal artifact volume compared with the 3D conventional imaging technique by 72% ± 23% for all evaluated image contrasts and both metal implants. The metal artifact volume differences were statistically significant when MSVAT-SPACE was compared with the 3D conventional technique. The blinded reads demonstrated that SEMAC-VAT and MSVAT-SPACE had distinctly superior quality compared with conventional acquisitions. Quality was measured in terms of artifact size, distortions, image quality and visualization of bone marrow and soft tissues adjacent to metal implants. This was the case for both tissue phantom images and human images with good interobserver agreement. Conclusions:SEMAC-VAT (2D) and MSVAT-SPACE (3D) demonstrated a consistent, marked reduction of metal artifacts for different metal implants and offered flexible image contrasts (T1, T2, PD and STIR) with high image quality. These techniques likely will improve the evaluation of postoperative patients with metal implants.

Rapid healing of femoral defects in rats with low dose sustained BMP2 expression from PEGDA hydrogel microspheres.

Current strategies for bone regeneration after traumatic injury often fail to provide adequate healing and integration. Here, we combined the poly (ethylene glycol) diacrylate (PEGDA) hydrogel with allogeneic “carrier” cells transduced with an adenovirus expressing BMP2. The system is unique in that the biomaterial encapsulates the cells, shielding them and thus suppressing destructive inflammatory processes. Using this system, complete healing of a 5 mm‐long femur defect in a rat model occurs in under 3 weeks, through secretion of 100‐fold lower levels of protein as compared to doses of recombinant BMP2 protein used in studies which lead to healing in 2–3 months. New bone formation was evaluated radiographically, histologically, and biomechanically at 2, 3, 6, 9, and 12 weeks after surgery. Rapid bone formation bridged the defect area and reliably integrated into the adjacent skeletal bone as early as 2 weeks. At 3 weeks, biomechanical analysis showed the new bone to possess 79% of torsional strength of the intact contralateral femur. Histological evaluation showed normal bone healing, with no infiltration of inflammatory cells with the bone being stable approximately 1 year later. We propose that these osteoinductive microspheres offer a more efficacious and safer clinical option over the use of rhBMP2.

Clearing the Cervical Spine in the Blunt Trauma Patient

The goal of cervical spine clearance is to establish that injuries are not present. Patients are classified into four groups: asymptomatic, temporarily nonassessable secondary to distracting injuries or intoxication, symptomatic, and obtunded. Level I evidence supports that the asymptomatic patient can be cleared on clinical grounds and does not require imaging. The temporarily nonassessable patient may have short‐term mental status changes (eg, intoxication, painful distracting injuries) and can be evaluated by two methods. When there is urgency, the evaluation is similar to that for the obtunded patient. Alternatively, the patient can be reevaluated within 24 to 48 hours, after return of mentation or following treatment of painful injuries. The patient then can be assessed as the asymptomatic patient is. The symptomatic patient requires advanced imaging. The obtunded patient should undergo, at minimum, a multidetector CT scan. Two methods are advocated. One uses only multidetector CT; a normal result is sufficient to clear the obtunded patient. The alternative method is obtaining a magnetic resonance image subsequent to a negative multidetector CT scan. Because at present information is insufficient to determine whether MRI is indicated, this is an area of controversy.