Dose-dependent effects of combined IGF-I and TGF-beta1 application in a sheep cervical spine fusion model.

Combined IGF-I and TGF-beta1 application by a poly-(D,L-lactide) (PDLLA) coated interbody cage has proven to promote spine fusion. The purpose of this study was to determine whether there is a dose-dependent effect of combined IGF-I and TGF-beta1 application on intervertebral bone matrix formation in a sheep cervical spine fusion model. Thirty-two sheep underwent C3/4 discectomy and fusion. Stabilisation was performed using a titanium cage coated with a PDLLA carrier including no growth factors in group 1 ( n=8), 75 micro g IGF-I plus 15 micro g TGF-beta1 in group 2 ( n=8), 150 micro g IGF-I plus 30 micro g TGF-beta1 in group 3 ( n=8) and 300 micro g IGF-I plus 60 micro g TGF-beta1 in group 4 ( n=8). Blood samples, body weight and temperature were analysed. Radiographic scans were performed pre- and postoperatively and after 1, 2, 4, 8, and 12 weeks. At the same time points, disc space height and intervertebral angle were measured. After 12 weeks, the animals were killed and fusion sites were evaluated using quantitative computed tomographic (CT) scans to assess bone mineral density, bone mineral content and bony callus volume. Biomechanical testing was performed and range of motion, and neutral and elastic zones were determined. Histomorphological and histomorphometrical analysis were carried out and polychrome sequential labelling was used to determine the time frame of new bone formation. In comparison to the group without growth factors (group 1), the medium- and high-dose growth factor groups (groups 3 and 4) demonstrated a significantly higher bony callus volume on CT scans, a higher biomechanical stability, an advanced interbody bone matrix formation in histomorphometrical analysis, and an earlier bone matrix formation on fluorochrome sequence labelling. Additionally, the medium- and high-dose growth factor groups (groups 3 and 4) demonstrated a significantly higher bony callus volume, a higher biomechanical stability in rotation, and an advanced interbody bone matrix formation in comparison to the low-dose growth factor group (group 2). No significant difference could be determined between the medium- and the high-dose growth factor groups (groups 3 and 4, respectively). The local application of IGF-I and TGF-beta1 by a PDLLA-coated cage significantly improved results of interbody bone matrix formation in a dose-dependent manner. The best dose-response relationship was achieved with the medium growth factor dose (150 micro g IGF-I and 30 micro g TGF-beta1). With an increasing dose of these growth factors, no further stimulation of bone matrix formation was observed. Although these results are encouraging, safety issues of combined IGF-I and TGF-beta1 application for spinal fusion still have to be addressed.

Comparison of BMP-2 and combined IGF-I/TGF-ss1 application in a sheep cervical spine fusion model.

Growth factors have proven to promote spine fusion. However, no comparative evaluation of growth factors in spinal fusion has yet been performed. The purpose of this study was to compare the efficacy and safety of combined IGF-I and TGF-ss1 application with BMP-2 application and autologous cancellous bone graft at an early time point in a sheep cervical spine fusion model. Thirty-two sheep underwent C3/4 discectomy and fusion. They were divided into four groups, according to their treatment: group 1, titanium cage ( n=8); group 2, titanium cage filled with autologous cancellous iliac crest bone grafts ( n=8); group 3, titanium cage coated with a poly-(D,L-lactide) (PDLLA) carrier including BMP-2 (5% w/w) ( n=8); group 4, titanium cage coated with a PDLLA carrier including IGF-I (5% w/w) and TGF-ss1 (1% w/w) ( n=8). Blood samples, body weight and temperature were analysed. Radiographic scans were performed pre- and postoperatively and after 1, 2, 4, 8 and 12 weeks. At the same time points, disc space height and intervertebral angle were measured. After 12 weeks, the animals were killed and fusion sites were evaluated using functional radiographic views in flexion and extension. Quantitative computed tomographic scans were performed to assess bone mineral density, bone mineral content and bony callus volume. Biomechanical testing was carried out and the values for range of motion, and neutral and elastic zone were determined. Histomorphological and histomorphometrical analysis were performed and polychrome sequential labelling was used to determine the time frame of new bone formation. The results showed that, in comparison to the group treated with the cage alone (group 1), the cage plus BMP-2 group (group 3) and the cage plus IGF-I and TGF-ss1 group (group 4) demonstrated a significantly higher fusion rate in radiographic findings, a higher biomechanical stability, a more advanced interbody fusion in histomorphometrical analysis, and an accelerated interbody fusion on fluorochrome sequence labelling. In comparison to the bone graft group (group 2), the BMP-2 (group 3) and IGF-I/TGF-ss1 group (group 4) showed significantly less residual motion on functional radiographic evaluation, higher bone mineral density of the callus and higher biomechanical stability in extension, rotation and bending. The BMP-2 group showed significantly less residual motion on functional radiographic evaluation and higher intervertebral bone matrix formation on fluorochrome sequence labelling at 9 weeks in comparison to the IGF-I/TGF-ss1 group. In contrast, the IGF-I/TGF-ss1 group showed a significantly higher bone mineral density of the callus than the BMP-2 group. In comparison to the autologous cancellous bone graft group, both growth factors (BMP-2 and combined IGF-I and TGF-ss1) significantly improved the biomechanical results of interbody fusion. No systemic side effects were observed for either growth factor. On the basis of these preliminary results, it would appear that combined IGF-I/TGF-ss1 application yields equivalent results to BMP-2 application at an early time point in anterior sheep cervical spine fusion.

[Experimental fusion of the sheep cervical spine. Part II: Effect of growth factors and carrier systems on interbody fusion]. / Experimentelle Spondylodese der Schafshalswirbelsäule Teil 2: Der Effekt von Wachstumsfaktoren und Carrier-Systemen auf die intervertebrale Fusion.

INTRODUCTION: A sheep cervical spine interbody fusion model was used to determine the effect of different carriers and growth factors on interbody bone matrix formation. The purpose of this study was to compare the efficacy and safety of combined IGF-I and TGF-beta1 application with BMP-2 application in spinal fusion. Additionally, a new poly (D, L-lactide) carrier system was compared to a collagen sponge carrier. METHOD: Forty sheep underwent C3/4 discectomy and fusion: group 1: titanium cage ( n=8), group 2: titanium cage coated with a PDLLA carrier (n=8), group 3: titanium cage coated with a PDLLA carrier including BMP-2 ( n=8), group 4: titanium cage with a collagen carrier including BMP-2 ( n=8), and group 5: titanium cage coated with a PDLLA carrier including IGF-I and TGF-beta1 ( n=8). Blood samples, body weight, and temperature were analyzed. Radiographic scans were performed pre- and postoperatively and after 1, 2, 4, 8, and 12 weeks, respectively. At the same time points, disc space height (DSH) and intervertebral angle (IVA) were measured. After 12 weeks the animals were killed and fusion sites were evaluated using functional radiographic views in flexion and extension. Quantitative computed tomographic scans (QCT) were performed to assess bone mineral density (BMD), bone mineral content (BMC), and bony callus volume (BCV). Biomechanical testing was carried out in flexion, extension, axial rotation, and lateral bending. Range of motion (ROM), neutral zone (NZ), and elastic zone (EZ) were determined. Histomorphological and histomorphometrical analyses were performed and polychrome sequential labeling was used to determine the time frame of new bone formation. RESULTS: In comparison to the non-coated cages, all PDLLA-coated cages showed significantly higher values for BMD of the callus and bone volume/total volume ratio. In comparison to the cage groups (groups 1 and 2), the cage plus BMP-2 (groups 3 and 4) and the cage plus IGF-I and TGF-beta1 group (group 5) demonstrated a significantly higher fusion rate in radiographic findings, a higher biomechanical stability, an advanced interbody fusion in histomorphometric analysis, and an accelerated interbody fusion on fluorochrome sequence labeling. BMP-2 application by the PDLLA carrier system (group 3) demonstrated significantly higher bony callus volume than BMP-2 application by a collagen sponge carrier (group 4). The BMP-2 group (group 3) showed significantly lower residual motion on functional radiographic evaluation and higher intervertebral bone matrix formation on fluorochrome sequence labeling at 9 weeks in comparison to the IGF-I/TGF-beta1 group (group 5). In contrast, the IGF-I/TGF-beta1 group (group 5) showed a significantly higher bone mineral density of the callus than the BMP-2 group (group 3). CONCLUSION: PDLLA coating of cervical spine interbody fusion cages as a delivery system for growth factors was effective and safe. In comparison to the collagen sponge carrier, the new PDLLA carrier system was able to improve results of interbody bone matrix formation. Both growth factors (BMP-2 and combined IGF-I and TGF-beta1) significantly accelerated results of interbody fusion. Based on these preliminary results, the combined IGF-I/TGF-beta1 application yields results equivalent to BMP-2 application at an early time in anterior sheep cervical spine fusion.

[Experimental fusion of the sheep cervical spine. Part I: Effect of cage design on interbody fusion]. / Experimentelle Spondylodese der Schafshalswirbelsäule Teil 1: Der Effekt des Cage-Designs auf die intervertebrale Fusion.

INTRODUCTION: There has been a rapid increase in the use of interbody fusion cages as an adjunct to spondylodesis, although experimental data are lacking. A sheep cervical spine interbody fusion model was used to determine the effect of different cage design parameters (endplate-implant contact area, maximum contiguous pore) on interbody fusion. MATERIAL AND METHOD: IN VITRO EVALUATION: 24 sheep cadaver specimens (C2-C5) were tested in flexion, extension, axial rotation, and lateral bending with a nondestructive flexibility method using a nonconstrained testing apparatus. Four different groups were examined: (1) control group (intact) ( n=24), (2) autologous tricortical iliac crest bone graft ( n=8), (3) Harms cage ( n=8), and (4) SynCage-C ( n=8). IN VIVO EVALUATION: 24 sheep underwent C3/4 discectomy and fusion: group 1: autologous tricortical iliac crest bone graft ( n=8), group 2: Harms cage filled with autologous cancellous iliac crest bone grafts ( n=8), and group 3: SynCage-C filled with autologous cancellous iliac crest bone grafts ( n=8). Radiographic scans were performed pre- and postoperatively and after 1, 2, 4, 8, and 12 weeks, respectively. At the same time points, disc space height (DSH), height index (HI), intervertebral angle (IVA), and endplate angle (EA) were measured. After 12 weeks the animals were killed and fusion sites were evaluated using biomechanical testing in flexion, extension, axial rotation, and lateral bending. Additionally, histomorphological and histomorphometrical analyses were performed. RESULTS: Over a 12-week period the cage groups showed significantly higher values for DSH, HI, IVA, and EA compared to the bone graft. In vivo stiffness was significantly higher for the tricortical iliac crest bone graft and Harms cage than in vitro stiffness. However, there was no difference between in vitro and in vivo stiffness of the SynCage-C. Histomorphometrical evaluation showed a more progressed bone matrix formation in the Harms cage group than in both other groups. CONCLUSION: The parameter endplate-implant contact area was not able to determine subsidence of cages. In contrast, the maximum contiguous pore of a cage significantly correlates with interbody bone matrix formation inside the cage. Additionally, there was no correlation between in vitro and in vivo stiffness of interbody fusion cages. Therefore, biomechanical in vitro studies are not able to determine in vivo outcome of fusion cages. Animal experimental evaluations of interbody fusion cages are essential prior to clinical use.

Incorporation of perforated and demineralized cortical bone allografts. Part I: radiographic and histologic evaluation.

Massive cortical bone allografts have been found to incorporate slowly into host bone. They are subject to complications such as nonunion, fatigue fracture and infection. In an attempt to improve osteoinduction in cortical bone allografts, laser perforated and partially demineralized cortical bone allografts were orthotopically transplanted into the sheep tibia. In this model, mid-shaft tibial bone allografts from out-bred sheep donor animals were prepared by partial demineralization and drilling of 0.33-mm diameter holes with a pulsed, 2.94-microm wavelength Erbium:Yttrium-Aluminum-Garnet laser. Recipient animals of the same out-bred strain were divided into three groups of eight according to the type of cortical allograft used: group 1, fresh-frozen, no treatment; group 2, laser hole grid; and group 3, laser hole grid and partial demineralization. Plain films were taken in two standard views at monthly intervals. Incorporation was evaluated at nine months postoperatively. Longitudinal radiographic data was correlated to a histologic and morphometric evaluation of each bone graft. Computer tomography was used for the latter analysis. Results showed that untreated allografts, although surrounded by a periosteal bone cuff, were poorly incorporated. Partial demineralization lead to excessive resorption of allografts, but little new bone formation. Laser perforation and partial demineralization induced complete incorporation of allografts into the host bone. Based on the results of the radiographic, histologic and morphometric evaluation, the development of laser-perforated and partially demineralized bone allografts was proposed for clinical use.

Incorporation of perforated and demineralized cortical bone allografts. Part II: A mechanical and histologic evaluation.

Laser perforated and partially demineralized cortical bone allografts were orthotopically transplanted into sheep tibiae. This paper reports results of the mechanical testing of the transplanted bones, which was done at nine months postoperatively. Animals were divided into three groups of eight according to the type of cortical allograft used: group 1, no treatment; group 2, laser hole grid; and group 3, laser hole grid and partial demineralization. Thus, changes in flexural rigidity of 24 transplanted whole tibiae were investigated. Starting in the anterior direction at the tibial tuberosity, the flexural rigidity was determined using a nondestructive 4-point bending test. The elliptical distribution of the flexural rigidity was compared to the untreated contralateral control bone of each animal. Mechanical parameters were defined as percentage rates for comparative analysis between groups. Flexural rigidity measurement showed that bones transplanted with untreated allografts were stiffer than contralateral control bones. Partial demineralization of allografts reduced the flexural rigidity of transplanted bones below the level of contralateral control bones. Flexural rigidities of test bones transplanted with laser perforated and partially demineralized allografts were higher than those seen in bones transplanted with partially demineralized allografts. These results were corroborated by the histologic analysis which showed that untreated allografts, although surrounded by a periosteal bone cuff that effectively increased their outer diameter. In contrast, excessive bone resorption was observed in partially demineralized allografts. Laser-perforated and partially demineralized allografts showed histologic evidence of complete incorporation into the host bone. Based on this mechanical evaluation, it was concluded that processing of cortical bone allografts by the combination of perforation and partial demineralization resulted in improved mechanical strength of the transplanted bones as compared to processing by partial demineralization alone.

Immune response to perforated and partially demineralized bone allografts.

Immune responses have been shown to be involved in the pathogenesis of clinical complications of cortical bone allografts. In an attempt to reduce the immunogenicity of these allografts, we evaluated cortical bone allografts modified by laser perforation and partial demineralization transplanted orthotopically into sheep tibiae. The recipient animals were divided into three groups, of eight animals each, according to the type of cortical allograft that was transplanted: group 1, no treatment (control); group 2, demineralization only; and group 3, laser perforation and partial demineralization. All animals were tissue-typed by biochemical definition of MHC class I molecules, using unidimensional isoelectric focusing and Western blotting. Mismatches of donors and recipients were assessed by testing samples of each donor and recipient pair in parallel and by comparing their individual bands. Donor-specific alloantibodies were detected by a similar technique, using an enzyme-linked immunosorbent assay (ELISA) format. Negative controls were included in all tests. All grafts were poorly immunogenic, whether they were untreated, processed by partial demineralization, or processed by both laser perforation and partial demineralization. Only two recipient animals showed a transient, antibody-mediated donor-specific immune response. One of these animals had received a control allograft, whereas the other animal had received a laser-perforated and partially demineralized bone allograft. All of the grafts in this study, including control grafts, were stripped of soft tissues and their bone marrow was removed; cellular sources of alloantibody stimulation may have been eliminated by these processes. The results of this study suggest that immune responses to bone allografts may be reduced by removing the bone marrow and adjacent soft tissues. The processing of cortical bone allografts by laser perforation and partial demineralization appeared to have little effect on immune responses.

Observations on fiber-forming collagens in the anulus fibrosus.

STUDY DESIGN: The spatial distribution of fiber-forming collagens in the anulus fibrosus was investigated in the complete longitudinal and horizontal sections of human lumbar intervertebral discs of seven individuals. OBJECTIVES: To obtain a more detailed structural definition of the anulus fibrosus because structural alterations of its collagen fiber network have been implicated in discal degeneration and other spinal pathologies. SUMMARY OF BACKGROUND DATA: Prior biochemical or immunofluorescence studies permitted only limited conclusions concerning the spatial distribution of the fiber-forming collagens in relation to anatomic structures because they were based on intraoperative tissue specimens or performed on incomplete sections of human intervertebral discs. METHODS: Complete human intervertebral discs with their adjacent vertebral bodies were fixed, decalcified, and embedded in paraffin. The intervertebral disc and its adjacent structures were reviewed in their entirety on one histologic slide. Monoclonal antibodies against human Types I, II, and III collagen were used for immunohistochemistry. A comparative analysis based on both immunohistochemical and histologic evaluation was performed. RESULTS: Type I collagen was seen abundantly in the outer zone and outer lamellas of the inner zone of the anulus fibrosus. On longitudinal sections, the Type I collagen distribution took the shape of a wedge. On horizontal sections, the Type I collagen positive area took the shape of a ring that was wider anteriorly than posteriorly. This suggests that the three-dimensional shape of the Type I collagen-positive tissue in the anulus fibrosus can be described by a donut that is wider anteriorly than posteriorly. Type II collagen was present in the entire inner of the anulus fibrosus, but not in the outer zone. In addition, it was found in the cartilaginous endplates. Type III collagen showed some codistribution with Type II collagen, particularly in pericellular locations in areas of spondylosis, which was noted at the endplates, vertebral rim, and insertion sites of the anulus fibrosus. CONCLUSIONS: These observations on the location of Types I and II collagen provide a more detailed structural definition of the anulus fibrosus, which may assist in further investigation of discal herniation.

Role of bone bark during growth in width of tubular bones. A study in human fetuses.

The morphologic features of bone bark, a structure surrounding the distal and proximal ends of long bones, were studied in the distal femur, proximal tibia, and proximal fibula of 77 spontaneously aborted human fetuses varying in gestational age from 10 to 20 weeks. Standard histologic techniques used in addition to in situ immunohistochemical staining allowed the examination of the structure of the bone bark and localization of Types 1, 2, and 3 collagens at different gestational ages. The bone bark was shaped like a cylindrical sheath of bone lamellae of varying thickness. The epiphyseal end of the bone bark, known as the groove of Ranvier, was covered outwardly by a fibrous layer and inwardly by the epiphyseal cartilage and contained mesenchymal cells, chondroblastic precursor cells, and densely packed cells differentiating into osteoblasts. Neither the cell density in the groove nor the thickness of the bone bark were identical circumferentially, indicating an unequal growth in width. In addition, the presence of periosteal apposition and endosteal resorption of the bone bark on one side and of endosteal bone deposition accompanied by periosteal resorption of the bone bark on the opposite side support the concept of a spatial drift of bones. These observations furnish histologic proof that groove and bone bark, although assuring an equal growth in length, contribute to an unequal and eccentric growth in width.

Concomitant meniscal and articular cartilage lesions in the femorotibial joint.

The frequency of concomitant meniscal and articular cartilage lesions in the femorotibial joint was analyzed in a retrospective study of 1740 knee joints examined arthroscopically with the objective of determining possible correlations between the two knee joint abnormalities. Articular cartilage lesions were found in 81.4% (N = 1416) of femorotibial joints examined and meniscal derangements were noted in 72.8% (N = 1268). In the medial compartment, concomitance was noted in 76.3% (821 of 1076) on the femoral condyles and in 48.6% (523 of 1076) on the tibial plateau. In the lateral compartment, 43.1% (212 of 492) of the knees with deranged menisci had femoral and 55.1% (271 of 492) had tibial articular lesions. Medial meniscal lesions were more frequently associated with femoral and tibial chondral degeneration than lateral meniscal derangements (P < 0.001). Longitudinal, bucket-handle, and complex tears of the medial meniscus were significantly more often associated with articular cartilage damage than horizontal cleavage, flap, or radial tears. Degeneration of the meniscus was highly correlated with chondral destruction in both compartments. No cause-and-effect relationship could be established, but practical implications of these findings are discussed.

Structural and functional changes in the canine shoulder after cessation of immobilization.

The functional and structural consequences of remobilization of the glenohumeral joint after 12 weeks of immobilization were studied in 10 beagle dogs. The dogs were grouped in pairs: 2 were used as controls, and in 8 others 1 forelimb was immobilized. At the end of 12 weeks, 2 dogs in the experimental group were euthanized, as were the 2 dogs used as controls. The cast was removed from the other 6 dogs, and the remobilized animals were euthanized in pairs at the end of 4, 8, and 12 weeks. It was found that after 12 weeks of immobilization, the passive range of motion was markedly impaired, intraarticular pressure was raised during movements, and the filling volume of the joint cavity was reduced. Histologically, the capsule showed synovial lining hyperplasia and vascular proliferation in the wall. Immunohistochemical labeling for collagen Types I and III failed to show an increase of fibrous collagens in the capsular wall. Both the functional and structural changes were unaltered after 4 weeks of remobilization, but after 8 weeks they began to reverse, and they returned to normal levels after 12 weeks. Both functional and structural changes after 12 weeks of immobilization of the uninjured glenohumeral joint are reversible by remobilization. The collagen composition of the capsule seems unrelated to the degree of capsular contraction that occurs during 12 weeks of immobilization and subsequent remobilization.

Classification of articular cartilage lesions of the knee at arthroscopy.

Classification systems for articular cartilage lesions in the knee have been based primarily on the appearance and severity of damage to the articular cartilage. Each of these systems has deficiencies in the documentation of articular cartilage abnormalities. This article proposes a new classification system for articular cartilage lesions in the knee. This system incorporates multiple parameters such as appearance, depth of involvement, area, clinical stage, and location of the lesion. A four-sector-mapping system allowing precise documentation of the location of articular cartilage lesions in the femorotibial joint is proposed. One numerical category is used to assess severity of a chondral defect. The use of these categories allows the surgeon to accurately grade and locate damage to the articular cartilage and its relationship to other concomitant intra-articular abnormalities. The proposed classification system has proven advantageous in communicating complex findings.

Effects of immobilization on the capsule of the canine glenohumeral joint. A structural functional study.

The effects of immobilization on the function of the glenohumeral joint and its capsule were investigated in eight beagle dogs. One foreleg of each animal was immobilized in a spica cast, and the dogs were euthanized after 4, 8, 12, and 16 weeks of immobilization in groups of two. The contralateral limb and the forelegs of two normal beagles were used as controls. At the time of euthanasia, the range of motion, intraarticular filling volume during passive movements of the joint, and intraarticular filling volume to rupture the capsule were measured. The synovium, including the capsule and the subscapular bursa, were examined histologically. In the immobilized limb, there was progressive restriction in the range of motion with increases in intraarticular pressure, and the filling volume that was required to cause a rupture of the capsule diminished. Morphologically, the capsule and the subscapular bursa showed focal adhesions. Thus, it appeared that immobilization of these canine forelimbs produced changes in the glenohumeral joint that resembled those of frozen shoulder in humans.

The pathogenesis of club foot. A histomorphometric and immunohistochemical study of fetuses.

We studied 16 club feet and 27 normal feet from spontaneously aborted human fetuses in the second trimester of gestation and measured the length of the spring ligament, and the declination angle and size of the talus. We also studied the cellular characteristics of the spring ligament and the immunohistochemical features of the medial ankle ligaments using monoclonal antibodies against type-III collagen, desmin, vimentin, and smooth muscle actin. Histomorphometric results indicated that the talar deformity was not the primary lesion. Histological and immunohistochemical findings showed that the cells and collagen fibres of the medial ankle ligaments of club feet appeared to be the site of the earliest changes, in that they had lost their spatial orientation and had contracted. In severe club feet before the third trimester of gestation, myofibroblast-like cells seemed to create a disorder of the ligaments resembling fibromatosis. This led to contraction and resulted in typical club-foot deformity.

Analysis and synthesis of periodic optical resonant reflectors.

Periodic resonant reflectors consisting of a periodic succession of equally spaced, uncoated, identical, optical plates are analyzed both by a matrix method known from thin film theory and by conventional electrical theory of cascaded two-ports. Simple formulas are derived for the design of resonant reflectors according to given specifications. As an example the resulting formulas are applied to design a periodic resonant reflector for a ruby laser.