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Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 31 - 31
1 Sep 2012
Gawri R Mwale F Ouellet JA Steffen T Roughley PJ Antoniou J Haglund L
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Purpose

Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response.

Method

Seventy lumbar IVDs, from 14 individuals, were isolated within 24 h after death. Discs were prepared for organ culture by removing bony endplates but retaining cartilaginous endplates (CEP). Discs were cultured with no external load applied. The effects of glucose and FBS concentrations were evaluated. Dulbeccos Modified Eagle Media (DMEM) was supplemented with glucose, 4.5g/L or 1g/L, referred to as high and low (physiological) glucose, and FBS, 5% or 1%, referred to as high and low FBS, respectively. After a four week culture period, samples were taken across the disc using a 4 mm biopsy punch. Cell viability was analyzed using a live/dead fluorescence assay (Live/Dead, Invitrogen) and visualized by confocal microscopy. CEP discs were also placed in long term culture for four months, and cell viability was assessed. Western bolt analysis for the G1 domain of aggrecan was also performed to assess the effect of nutritional state on disc catabolism.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 14 - 14
1 Sep 2012
Han Y Sardar Z McGrail S Steffen T Martineau P
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Purpose

Twelve case reports of distal femur fractures as post-operative complications after anterior cruciate ligament (ACL) reconstruction have been described in the literature. The femoral tunnel has been suggested as a potential stress riser for fracture formation. The recent increase in double bundle ACL reconstructions may compound this risk. This is the first biomechanical study to examine the stress riser effect of the femoral tunnel(s) after ACL reconstruction. The hypotheses tested in this study are that the femoral tunnel acts as a stress riser to fracture and that this effect increases with the size of the tunnel (8mm versus 10mm) and with the number of tunnels (one versus two).

Method

Femoral tunnels simulating single bundle (SB) hamstring graft (8 mm), bone-patellar tendon-bone graft (10 mm), and double bundle (DB) ACL reconstruction (7mm, 6 mm) were drilled in fourth generation saw bones. These three experimental groups and a control group consisting of native saw bones without tunnels, were loaded to failure.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVIII | Pages 34 - 34
1 Sep 2012
Gawri R Mwale F Ouellet J Steffen T Roughley P Haglund L Antoniou J
Full Access

Purpose

Disc degeneration is known to occur early in adult life, but at present there is no medical treatment to reverse or even retard the problem. Development of medical treatments is complicated by the lack of a validated long term organ culture model in which therapeutic candidates can be studied. The objective of this study was to optimize and validate an organ culture system for intact human intervertebral disc (IVD), which could be used subsequently to determine whether synthetic peptide growth factors can stimulate disc cell metabolism and initiate a repair response.

Method

Seventy lumbar IVDs, from 14 individuals, were isolated within 24 h after death. Discs were prepared for organ culture by removing bony endplates but retaining cartilaginous endplates (CEP). Discs were cultured with no external load applied. The effects of glucose and FBS concentrations were evaluated. Dulbeccos Modified Eagle Media (DMEM) was supplemented with glucose, 4.5g/L or 1g/L, referred to as high and low (physiological) glucose, and FBS, 5% or 1%, referred to as high and low FBS, respectively. After a four week culture period, samples were taken across the disc using a 4 mm biopsy punch. Cell viability was analyzed using a live/dead fluorescence assay (Live/Dead, Invitrogen) and visualized by confocal microscopy. CEP discs were also placed in long term culture for four months, and cell viability was assessed. Western bolt analysis for the G1 domain of aggrecan was also performed to assess the effect of nutritional state on disc catabolism.


Orthopaedic Proceedings
Vol. 94-B, Issue SUPP_XXXVI | Pages 85 - 85
1 Aug 2012
Steffen T Freeman B Aebi M
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Long term, secondary implant fixation of Total Disc Replacements (TDR) can be enhanced by hydroxyapatite or similar osseo-conductive coatings. These coatings are routinely applied to metal substrates. The objective of this in vivo study was to investigate the early stability and subsequent bone response adjacent to an all polymer TDR implant over a period of six months in an animal model.

Six skeletally mature male baboons (Papio annubis) were followed for a period of 6 months. Using a transperitoneal exposure, a custom-sized Cadisc L device was implanted into the disc space one level above the lumbo-sacral junction in all subjects. Radiographs of the lumbar spine were acquired prior to surgery, and post-operatively at intervals up to 6 months to assess implant stability. Flourochrome markers (which contain molecules that bind to mineralization fronts) were injected at specified intervals in order to investigate bone remodeling with time.

Animals were humanely euthanized six months after index surgery. Test and control specimens were retrieved, fixed and subjected to histological processing to assess the bone-implant-bone interface. Fluorescence microscopy and confocal scanning laser microscopy were utilized with BioQuant image analysis to determine the bone mineral apposition rates and gross morphology.

Radiographic evaluation revealed no loss of disc height at the operative level or adjacent levels. No evidence of subsidence or significant migration of the implant up to 6 months. Heterotopic ossification was observed to varying degrees at the operated level.

Histology revealed the implant primary fixation features embedded within the adjacent vertebral endplates. Flourochrome distribution revealed active bone remodeling occurring adjacent to the polymeric end-plate with no evidence of adverse biological responses. Mineral apposition rates of between 0.7 and 1.7 microns / day are in keeping with literature values for hydroxyapatite coated implants in cancellous sites of various species.

Radiographic assessment demonstrates that the Cadisc L implant remains stable in vivo with no evidence of subsidence or significant migration. Histological analysis suggests the primary fixation features are engaged, and in close apposition with the adjacent vertebral bone. Flourochrome markers provide evidence of a positive bone remodelling response in the presence of the implant.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 48 - 48
1 Mar 2008
Morin P Reindl R Steffen T Harvey E Guy P
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In our cadaver study plating the fibula in addition to nailing the tibia decreased the mean rotation across the tibial osteotomy site compared to nailing the tibia alone. Although this is statistically significant (p=0.0034) it may not be clinically relevant as the mean values for ROM were 19.10 and 17.96 degrees respectively. Plating the fibula resulted in no statistically significant difference in the mean vertical displacement, angulation or neutral zone.

Therefore, we may conclude that plating the fibula in a combined distal third tibia and fibula fracture does not enhance the stability of tibial IM nailing.

The purpose of this study was to determine if combined distal third tibia and fibula fractures are more stable when fibular fixation is added to the standard tibial IM rodding.

In combined distal third tibia and fibula fractures, plating the fibula does not enhance stability of intramedullary tibial nailing.

No additional incision or soft tissue stripping is required for plating of the fractured fibula.

The average range of motion in rotation was 19.1° for tibial and fibular fixation combined, and 18.0 ° for tibial fixation alone with a difference of 1.1°, which was clinically significant ( p=0.0034). The mean differences in vertical displacement, angulation, and neutral zone were not statistically significant.

Five matched pairs of embalmed cadaveric lower limbs were dissected and stripped of soft tissue. Each tibia received a 9mm solid titanium nail that was locked proximally and distally. Fibular fixation consisted of a seven- hole LCDCP. A 1.5 cm section of tibia and a 1.0 cm section of fibula were removed. Testing was accomplished with an MTS machine. Vertical displacement was tested with an axial load to 500 N, rotation was tested with an internal and external torque of 5 Nm, and angulation was calculated from the vertical displacement data. All displacement data was measured across the osteotomy site.

The mean range of motion in rotation was the only statistically significant finding. However, considering the average range of motion with and without fibular plating of 17.96° and 19.10° respectively, this finding is likely not clinically relevant.

Funding: Tibial nails, bolts, fibular plates and screws provided by Synthes (Paoli, PA, USA)


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 91 - 91
1 Mar 2008
Walsh S Reindl R Harvey E Berry G Beckman L Steffen T
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Many two-part fractures of the proximal humerus are treated conservatively due to the frequent failure of internal fixation. The current investigation examines the biomechanical properties of a unique plate versus a standard plate for internal fixation of proximal humerus fractures. The unique plate employs screws that thread into the plate, creating a multi-planer, fixed angle device. A cadaveric model was developed that relied on the rotator cuff musculature as the primary deforming force. The locking plate displayed significantly greater holding power on the humeral head (p=0.007). This may lead to more consistent results in two-part fractures treated with internal fixation.

The current investigation evaluates a unique plate designed to treat fractures of the proximal humerus. The plate employs screws that thread into the plate creating a fixed angle device. This plate was tested versus a standard cloverleaf plate.

The locking plate displayed greater holding power on the humeral head in the model tested. This may lead to decreased failure rates in two-part fractures treated with internal fixation.

Many two-part fractures of the proximal humerus are treated conservatively due to the frequent failure of common internal fixation modalities. This is done with the acceptance of possible non-union and loss of function. A more reliable method for stable internal fixation is therefore desirable.

A significant difference was found (p=0.007) with the locking plate displaying greater holding capability on the humeral head.

Eight pairs of preserved, cadaveric humeri were dissected and plated with either the locking plate or standard cloverleaf plate followed by an osteotomy at the surgical neck. A servo-hydraulic testing machine was then used to pull on the rotator cuff musculature until failure was achieved. Failure of the plate-head interface was reached in five of the eight pairs. Previous biomechanical studies have not taken into account the clinical mode of failure when testing internal fixation modalities for proximal humerus fractures. The current study has reproduced failure into varus by relying on the rotator cuff musculature as the primary deforming force.

Funding: All implants donated by Synthes, Canada


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 72 - 72
1 Mar 2008
Morin P Reindl R Steffen T Ouellet J Arlet V Aebi M
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“Delta fixation” was developed to treat low-grade L5 isthmic spondylolisthesis. It involves placement of pedicle screws into L5 and from S1 through the L5/S1 disc into L5 (Fig.1). A biomechanical comparison to standard Posterior Lumbar Interbody Fusion (PLIF) with two anterior cages and pedicle screws in L5 and S1 was made. Eight fresh frozen human specimens were instrumented with both fixations and tested. Delta fixation was significantly more stable in resisting rotation. It requires less manipulation of the nerve roots and spares the cost of the fusion cages.

Our objective is to compare the stability of Delta versus PLIF fixation in the treatment of low grade isthmic spondylolisthesis

Delta fixation provides superior initial stability, and therefore is an acceptable alternative to PLIF for the treatment of low grade isthmic spondylolisthesis of L5-S1.

Symptomatic low-grade isthmic spondylolisthesis of L5 is often managed with PLIF. This procedure requires extensive manipulation of the cauda equine, posterior resection of the disc and the placement of two inter-body cages as spacers in addition to pedicle screws in L5 and S1.

Delta Fixation has been developed to provide stable fixation with less nerve root manipulation and without the use of inter-body cages. It is therefore a safer alternative method of fixation that spares the additional cost of the fusion cages.

When comparing Delta fixation to PLIF fixation the only statistically significant difference was found in axial rotation. Delta fixation had 2.05 degrees less ROM and 0.90 degrees less NZ compared to PLIF fixation with P values of 0.0052 and 0.0104 respectively. This demonstrates that the delta fixation is more stable than PLIF fixation.

Eight fresh frozen human spines were used. Matched pairs were created and block randomization used to create two groups: PLIF fixation and Delta fixation groups. The specimens were instrumented with a grade II spondylolisthesis of L5-S1, tested, and then re-instrumented with the alternative fixation and tested again. Vertical displacement, axial rotation, flexion– extension and side bending were tested using an MTS machine.

Please contact author for tables and/or diagrams.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 40 - 40
1 Mar 2008
Antoniou J Roughley P Aebi M Steffen T Mwale F
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Hyaline cartilage and immature nucleus pulposus possess similar macromolecules in their extracellular matrix, and there is no unique molecular marker to distinguish the two tissues. We show that in normal disc (fifteen to twenty-five years old), the GAG to hydroxyproline ratio (proteoglycan to collagen ratio) within the nucleus pulposus is approximately 28:1. However, the GAG to hydroxyproline ratio within hyaline cartilage of the same group is 2.5:1. This information is important in identifying stem cell conversion to a nucleus pulposus cell phenotype rather than a chondrocyte phenotype for tissue engineering of intervertebral disc.

Tissue engineering of intervertebral discs (IVDs) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. Since there is no unique marker for disc tissue, and because cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. In this study, we compare the proteoglycan to collagen ratio in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals.

To distinguish between a normal NP and hyaline cartilage phenotype for tissue engineering of IVDs.

Human lumbar spine specimens were harvested from fresh cadavers, aged twelve week to seventy-nine year. Discs and endplates were examined for total collagen using the hydroxyproline assay and glycosaminoglycan (GAG) content using a standard assay.

In a mature disc with no degeneration (fifteen to twentyfive years), the GAG to hydroxyproline ratio within the NP is approximately 28:1. However, the ratio within the hyaline cartilage endplate of the same group is 2.5:1.

A high proteoglycan to collagen ratio can be used to distinguish NP cells from chondrocytes. The lower NP collagen content is probably responsible for its gelatinous nature rather than the firm texture of hyaline cartilage, and this is essential for normal disc function. This information is crucial in identifying a NP-like phenotype when MSCs are induced to differentiate into a disc cell as opposed to a chondrocyte, for tissue engineering of IVDs.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 106 - 106
1 Mar 2008
Martineau P Bergeron S Beckman L Steffen T Harvey EJ
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Radial-sided avulsions of the TFCC (Palmer 1d) remain a challenging pathology to treat. No current procedures have addressed these injuries successfully and reproducibly. Ten preserved dissected cadaveric forearm specimens with intact TFCC and without ulnar positive variance underwent biomechanical testing. Specimens were tested intact, then with Palmer 1d TFCC lesion and finally post-reconstruction. Measurement of total displacement with a −20N to 20N load was performed. The results indicate that our novel anatomic intra-articular reconstruction of unstable radial-sided TFCC avulsions was successful in restoring baseline stability to the DRUJ without interfering with pronation or supination.

Radial-sided avulsions of the TFCC (Palmer 1d) remain a challenging pathology to treat. No current procedures have addressed these injuries successfully and reproducibly. We tested a novel intra-articular reconstruction to address unstable radial-sided TFCC avulsions.

Ten preserved dissected cadaveric forearm specimens with intact TFCC and without ulnar positive variance underwent biomechanical testing using an MTS machine. Measurement of total displacement with a −20N to 20N load was performed. Specimens were tested intact, then with Palmer 1d TFCC lesion and finally post-reconstruction. All tests were performed at neutral, maximal pronation and maximal supination.

Mean total displacements of the specimens at neutral rotation were: 4.122mm ± 0.363 for the intact specimens compared to 11.839mm ± 0.782 after creation of the tear (p< 0.000002) and 3.883mm ± 0.655 for the reconstructed specimens (p=0.77). In maximal pronation mean total displacements were: 2.378mm ± 0.250 intact vs. 4.922 ± 0.657 torn (p< 0.0007) and 2.124mm ± 0.339 post-reconstruction (p=0.61). In maximal supination mean total displacements were: 1.438mm ± 0.222 intact vs. 5.704mm ± 1.258 torn (p< 0.006) and 1.004mm ± 0.091 post-reconstruction (p=0.07). All specimens obtained the same maximal pronation and supination pre and post-reconstruction.

Restoration of stability and joint function have never been achieved with previous reconstruction attempts of radial-sided TFCC avulsions. Current procedures are unable to restore DRUJ stability without a significant sacrifice of motion. Our anatomic intra-articular reconstruction of unstable radial-sided TFCC avulsions succeeded in restoring baseline stability to the DRUJ without interfering with pronation/supination.


Orthopaedic Proceedings
Vol. 90-B, Issue SUPP_I | Pages 134 - 134
1 Mar 2008
Ciobanu I Mwale F Giannitsios D Roughley P Steffen T Antoniou J
Full Access

Purpose: In vivo, intertervertebral disc cells exist in a low oxygen environment ranging from 5% O2 for the annulus fibrosus (AF) cells to 1% O2 for the nucleus pulpous (NP) cells. Various conditions have been used for in vitro cell culture and seem that AF and NP cells can respond differently in the different systems, which may differ from the in vivo environment in terms of nutrient supply, O2 levels and biomechanical loading. The aim of this study was to determine how AF and NP cells respond to different O2 concentrations when cultured in a 3 dimensional system consisting of an alginate scaffold.

Methods: Bovine AF and NP cells were embedded in alginate beads and incubated in airtight polypropylene containers at different O2 concentration of 1%, 5% or 21%. Culture medium was changed every third day and the culture was carried out for 21 days. The pro-teoglycan content of the medium was analyzed using the DMMB assay. Cells were recovered from the alginate beads at two time points, day 8 and day 21 and RT-PCR was performed to amplify gene expression of GAPDH and aggrecan.

Results: In both cell types, the cumulative production of GAG increased with time in culture up to day 9, and then tended to plateau in the AF cells but continue to increase in the NP cells. At all time points, the level of GAG synthesis by NP cells was greater than by AF cells. All GAG synthesis trends were the similar at all O2 levels (1%, 5% and 21%).

Conclusions: In the alginate scaffold NP cells continue to exhibit their in situ behaviour by producing more proteoglycan than AF cells. Perhaps surprisingly, both cell types showed little change in GAG production with variations of O2 levels from 1–21%. This contrasts with other studies where GAG production is dependent on O2 concentrations. In the culture system used in this work, both cell types metabolize easily well at low oxygen as they do in normal conditions. Funding: Other Education Grant


Orthopaedic Proceedings
Vol. 84-B, Issue SUPP_I | Pages - 46
1 Mar 2002
Papin P Steffen T Reindle R Olah A Arvite T Stoll T Bohner M Aebi M
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Purpose: A preliminary biomechanical test conducted on cadaver specimens validated a new technique for vertebral bone harvesting for anterior intervertebral grafting of the lumbar spine. A cylinder of autologous bone harvested from a neighboring vertebra was used for the intervertebralimplant. The harvesting site was filled with a bone substitute. The biomechanical tests confirmed good restoration of the vertebral body structure. An in vivo study was conducted in the baboon. A block of tricalcium-phosphate (beta-TCP) impregnated with transforming growth factor beta3 (TGF-beta3) was used to fill the bone gap. The purpose of the present study was: 1) to assess the efficacy of this in vivo technique on a primate model, 2) to validate the surgical technique.

Material and methods: The retroperitoneal approach was used to operate nine baboons. Eighteen bone cylinders were harvested. The harvesting hole was left empty or filled with a 15 mm diameter beta-TCP cylinder, or with a beta-TCP cylinder impregnated with TGR-beta3. Control scans were obtained at three and six months postoperatively. The baboons were sacrificed at 6 months and the vertebral bodies were removed for histology study.

Results: There was no evidence of fracture or loss of vertebral body height. The harvesting holes left empty did not fill, while osteointegration and substantial resorption of the bone substitute was observed in the two other groups. In the group with beta-TCP impregnated with TGF-beta3 the resorption of the cylinder was more complete and signs of prevertebral neoformation of subperiosteal bone, not observed in the beta-TCP group, was observed. There was no neoformation of bone in the spinal canal or in the foramens. The scans showed progressive resorption starting three months postoperatively in the beta-TCP group impregnated with TGF-beta3.

Conclusion: The beta-TCP block used is a very good bone substitute for the primate spine. TGF-beta3 accelerates bone resorption and induces neoformation of subperiosteal bone. The new surgical technique for local harvesting of vertebral bone was validated.