Osteogenic behavior of alginate encapsulated bone marrow stromal cells: an in vitro study.

Sodium alginate is a useful polymer for the encapsulation and immobilization of a variety of cells in tissue engineering because it is biocompatible, biodegradable and easy to process into injectable microbeads. Despite these properties, little is known of the efficacy of calcium cross-linked alginate gel beads as a biodegradable scaffold for osteogenic cell proliferation and differentiation. In this study, we investigated the ability of rabbit derived bone marrow cells (BMCs) to proliferate and differentiate in alginate microbeads and compared them with BMCs cultured in poly-L-lysine (PLL) coated microbeads and on conventional 2D plastic surfaces. Results show that levels of proliferation and differentiation in microbeads and on tissue culture plastics were comparable. Cell proliferation in microbeads however diminished after fortification with a coating layer of PLL. Maximum cell numbers observed were, 3.32 x 10(5) +/- 1.72 x 103; 3.11 x 10(5) +/- 1.52 x 10(3) and 3.28 x 10(5) +/- 1.21 x 10(3 ) for the uncoated, PLL coated and plastic surface groups respectively. Alkaline phosphatase and protein expressions reflected the stage of cell differentiation. We conclude that calcium cross-linked alginate microbeads can act as a scaffold for BMC proliferation and osteogenic differentiation and has potential for use as 3D degradable scaffold.

Direct internal kyphectomy for severe angular tuberculous kyphosis.

We describe a direct internal kyphectomy through a modified costotransversectomy, an extrapleural approach to the kyphus that does not jeopardize already compromised pulmonary function. A curved longitudinal incision is made 6 to 8 cm lateral to the midline. The posterior 5 cm of the two to three crowded ribs at the apex are resected. The segmental intercostal nerves are preserved as a guide into the spinal canal. Two to three pedicles at the apex are resected. The pleura are elevated with blunt dissection leading to the internal kyphus. Removal of the posterior half of the collapsed vertebrae is performed with a high-speed burr; the posterior walls are removed last to avoid forward migration of the dural sac as the decompression progresses. Cortical strut grafting is then performed as far anteriorly as the exposure permits. We treated five patients with paraparesis of healed disease with this approach. Preoperatively the mean kyphosis was 114 degrees. Neurological improvement was obtained in two patients. At a mean followup of 5 years, solid anterior fusion was achieved in four patients. One patient died 5 months after surgery because of chest infection.

Phenotypic and population differences in the association between CILP and lumbar disc disease.

BACKGROUND: Lumbar disc disease (LDD) is one of the leading causes of disability in the working-age population. A functional single-nucleotide polymorphism (SNP), +1184T-->C, in exon 8 of the cartilage intermediate layer protein gene (CILP) was recently identified as a risk factor for LDD in the Japanese population (odds ratio (OR) 1.61, 95% CI 1.31 to 1.98), with implications for impaired transforming growth factorbeta1 signalling. AIM: To validate this finding in two different ethnic cohorts with LDD. METHODS: This SNP and flanking SNPs were analysed in 243 Finnish patients with symptoms of LDD and 259 controls, and in 348 Chinese subjects with MRI-defined LDD and 343 controls. RESULTS AND CONCLUSION: The results showed no evidence of association in the Finnish (OR = 1.35, 95% CI 0.97 to 1.87; p = 0.14) or the Chinese (OR = 1.05, 95% CI 0.77 to 1.43; p = 0.71) samples, suggesting that cartilage intermediate layer protein gene is not a major risk factor for symptoms of LDD in Caucasians or in the general population that included individuals with or without symptoms.

In vitro evaluation of alginate encapsulated adipose-tissue stromal cells for use as injectable bone graft substitute.

This study aims to investigate the survival and osteogenic behavior of murine-derived adipose-tissue stromal cells (ATSCs) encapsulated in alginate microcapsules thereby instigating further studies in this cell delivery strategy for in vivo osteogenesis. Cell viability was quantified using a tetrazolium-based assay and osteogenic differentiation was evaluated by both alkaline-phosphatase (ALP) histochemistry and osteocalcin mRNA analysis. Following microencapsulation, cell numbers increased from 3.9 x 10(3) on day 1 to 7.8 x 10(3) on day 7 and maintained excellent viability in the course of 21-day culture. ALP was 6.9, 5.5, and 3.2 times higher than monolayer cultures on days 7, 14, and 21, respectively. In addition, osteocalcin mRNA was detectable in encapsulated cultures earlier (day 14) than monolayer cultures. We conclude that alginate microcapsules can act as three-dimensional matrix for ATSC proliferation and has potential for use as injectable, biodegradable scaffold in bone tissue engineering.

Cervical intervertebral disc degeneration induced by unbalanced dynamic and static forces: a novel in vivo rat model.

STUDY DESIGN: Establishment of a novel in vivo animal model of cervical spondylosis. OBJECTIVE: To investigate apoptotic, degenerative, and inflammatory changes occurring in the cervical intervertebral discs of rats. SUMMARY OF BACKGROUND DATA: Cervical degeneration occurs as the result of imbalance of both static and dynamic spinal stabilizers. The disc degeneration that occurs is characterized by increased local inflammation and increased apoptosis of intervertebral disc cells. METHODS: By excising the paraspinal musculature and posterior cervical spinal ligaments of rats, both static and dynamic cervical stabilizers were disrupted. The resultant biomechanical imbalance resulted in biochemical and histologic changes, which were characterized by light microscopy, electron microscopy, immunostaining, enzyme-linked immunosorbent assay, polymerase chain reaction, and in situ hybridization. RESULTS: Histologic analysis showed characteristic degenerative changes of the intervertebral discs and vertebral endplates following surgery. Ultrastructural examination revealed apoptotic changes, which were verified by immunostaining. Instability also resulted in significant up-regulation of inflammatory factors, as shown by enzyme-linked immunosorbent assay, polymerase chain reaction, and in situ hybridization. CONCLUSIONS: By creating static and dynamic posterior instability of the cervical spine, this novel model of cervical spondylosis results in rapid intervertebral disc degeneration characterized by increased apoptosis and local inflammation, such as that seen clinically.

Biomechanical and electromyographic evaluation of ankle foot orthosis and dynamic ankle foot orthosis in spastic cerebral palsy.

This study evaluated the biomechanical and electromyographic effects of conventional ankle foot orthoses (AFOs) and dynamic ankle foot orthoses (DAFOs) on gait in patients with spastic cerebral palsy (CP). Thirteen patients with dynamic equinus underwent motion analysis with electromyography. Both AFOs and DAFOs provided longer stride length, permitted pre-positioning for initial contact, and successfully controlled the excessive plantarflexion during the swing phase. Median frequency (MF) of EMG signal indicated that extremely high firing was found in the patient's lower limbs compared to controls that resulted in tiredness. The DAFOs allowed a significantly larger total ankle range of motion than the AFOs. However, AFOs significantly reduced the MF while DAFOs did not. The reduced MF seen when wearing AFOs suggested an improvement of walking endurance. The DAFO had the advantage of less restriction on ankle movement, which avoids muscular atrophy and improves orthotic compliance.

Ultrastructural study of mineralization of a strontium-containing hydroxyapatite (Sr-HA) cement in vivo.

The purpose of this study was to investigate the mineralization leading to osseointegration of strontium-containing hydroxyapatite (Sr-HA) bioactive bone cement injected into cancellous bone in vivo. Sr-HA cement was injected into the ilium of rabbits for 1, 3, and 6 months. The bone mineralization area was found to be largest at 3 months, then at 1 month, and smallest at 6 months (p < 0.01) measured with tetracycline labeling. Osseointegration of Sr-HA cement was achieved at 3 months as observed by scanning electron microscopy. A high calcium and phosphorus area was observed at the interface of bone-Sr-HA cement determined by energy-dispersive X-ray analysis. Transmission electron microscopy gave evidence of the mechanism of bone formation. Dissolution of Sr-HA into debris by the bone remodeling process was thought to increase the concentration of calcium and phosphorus at the interface of bone-Sr-HA cement and stimulate bone formation. Crystalline Sr-HA formed an amorphous layer and dissolved into the surrounding solution, then apatite crystallites were precipitated and formed new bone at 3 months. This young bone then becomes mature bone, which bonds tightly to the Sr-HA cement with collagen fibers inserted perpendicularly after 6 months.

Microfracture and changes in energy absorption to fracture of young vertebral cancellous bone following physiological fatigue loading.

STUDY DESIGN: Fifty-five human thoracolumbar vertebrae were randomly fatigue loaded and analyzed. OBJECTIVES: The purpose of this study was to explore the relationship between fatigue loading, trabecular microfracture, and energy absorption to fracture in human cadaveric thoracolumbar vertebrae. BACKGROUND: Although trabecular microfractures are found in vivo and have been produced by fatigue loading in vitro, the effect of the level of physiologic fatigue loading on microfracture and energy absorption has not been investigated. METHODS: Fifty-five human thoracolumbar vertebrae (T11-L4) were randomly divided into 5 groups: 1) control (no loading, n = 6); 2) axial compression to yield (n = 7); and 3-5) 20,000 cycles of fatigue loading at 2 Hz (each n = 14). The level of fatigue loading was determined as a proportion of the yield load of Group 2 as follows: 10% (Group 3), 20% (Group 4), and 30% (Group 5). Half of the specimens in groups 3 to 5 were used for radiographic and histomorphometric analysis to determine microfracture density and distribution, whereas the other half were tested to determine the energy absorption to yield failure. RESULTS: No radiographic evidence of gross fracture was found in any of the groups following fatigue loading. A mean 7.5% increase in stiffness was found in specimens subject to cyclic loading at 10% of yield stress (Group 3). Fatigue at 20% (Group 4) and 30% of yield stress (Group 5) caused significantly higher (P < 0.05) increases in mean stiffness of 23.6% and 24.2%, respectively. Microfracture density increased from 0.46/mm in Group 3 to 0.66/mm in Group 4 and 0.94/mm in Group 5 (P < 0.05). The energy absorbed to failure decreased from 21.9 J in Group 3 to 18.1 J and 19.6 J in Groups 4 and 5, respectively (P < 0.05). CONCLUSIONS: Fatigue loading at physiologic levels produced microfractures that are not detectable by radiography. Increased fatigue load results in an increase in microfracture density and decrease energy absorbed to fracture, indicating a reduced resistance to further fatigue loading.

Strengthening mechanisms of bone bonding to crystalline hydroxyapatite in vivo.

The formation and strengthening mechanisms of bone bonding of crystalline hydroxyapatite (HA) has been investigated using high-resolution transmission electron microscope (HRTEM) and energy-dispersive X-ray (EDX) analysis. A series of results were obtained: (i) a layer of amorphous HA, which has almost the same chemistry as the implanted HA, was formed on the surface of crystalline HA particles prior to dissolution; (ii) at 3 months a bone-like tissue formed a bonding zone between mature bone and the HA implant, composed of nanocrystalline and amorphous apatite; and (iii) at 6 months, mature bone was in direct contact with HA particles, and collagen fibres were perpendicularly inserted into the surface layer of implanted HA crystals. Findings (i) and (ii) indicated the following dissolution-precipitation process. (i) The crystalline HA transforms into amorphous HA; (ii) the amorphous HA dissolves into the surrounding solution, resulting in over-saturation; and (iii) the nanocrystallites are precipitated from the over-saturated solution in the presence of collagen fibres. A preliminary analysis indicated several conclusions: (i) the transition from crystalline to amorphous HA might be the controlling step in the bone bonding of crystalline HA; (ii) biological interdigitation (or incorporation) of collagen fibres with HA and chemical bonding of a apatite layer were both necessary to strengthen and toughen a bone bond, not only for the bonding between bone and HA at 6 months, but also for the bonding zone at 3 months, which would otherwise be very fragile due to the inherited brittleness of polycrystalline ceramics; and (iii) perpendicular interdigitation is an effective way for collagen fibres to impart their unique combination of flexibility and strength to the interface which they are keying.

Surface treatment of injectable strontium-containing bioactive bone cement for vertebroplasty.

A novel injectable bioactive bone-bonding cement (SrHAC) composed of strontium-containing hydroxyapatite (Sr-HA) as the inorganic filler and bisphenol A diglycidylether dimethacrylate (Bis-GMA) as the organic matrix for vertebroplasty was developed previously. In this study, the Sr-HA powders were surface treated with methyl methacrylate (MMA) to improve the interface integration of the two phases. After surface treatment, the compression strength and Young's modulus, which were tested after immersion in distilled water at 37 degrees C for 24 h according to ISO 5833, were increased by 68.65 % (p <.001) and 31.02% (p <.001), respectively. The bending strength and bending stiffness of the bioactive bone cement were significantly improved by 54.44% (p <.001) and 83.90% (p <.001). In addition, the handling property of the cement was also enhanced. In vitro biomechanical testing showed that the stiffness of the fractured spine recovered to 82.5% (p <.01) of the intact condition after cementation with surface-treated SrHAC. The failure load of the spine cemented with original and MMA-treated SrHAC improved by 14.25% (p <.05) and 46.91% (p <.05) in comparison with the fractured spines. Results from this study revealed that the MMA-treated SrHAC has a better mechanical effect for orthopedic applications.

In vivo cancellous bone remodeling on a strontium-containing hydroxyapatite (sr-HA) bioactive cement.

The purpose of this study was to investigate the in vivo bone response to the strontium-containing hydroxyapatite (Sr-HA) bioactive bone cement injected into the cancellous bone. Sr-HA cement was injected into the iliac crest of rabbits for 1, 3, and 6 months. Active bone formation and remodeling were observed after 1 month. Newly formed bone was observed to grow onto the bone cement after 3 months. Thick osteoid layer with osteoblasts formed along the bone and guided over the bone cement surface reflected the stimulating effect of Sr-HA. From scanning electron microscopy (SEM) and energy-dispersive X-ray (EDX) analysis, high calcium and phosphorus levels were detected at the interface with a thick layer of 70 microm in width, and fusion of Sr-HA with the bone was observed. Blood vessels were found developing in remodeling sites. The affinity of bone on Sr-HA cement was increased from 73.55 +/- 3.50% after 3 months up to 85.15 +/- 2.74% after 6 months (p < 0.01). In contrast to Sr-HA cement, poly(methyl methacrylate) (PMMA) bone cement was neither osteoconductive nor bioresorbable. Results show that the Sr-HA cement is biocompatible and osteoconductive, which is suitable for use in treating osteoporotic vertebral fractures.

Spinal flexibility increase after chymopapain injection is dose dependent: a possible alternative to anterior release in scoliosis.

STUDY DESIGN: Experimental animal study. OBJECTIVES: To investigate whether the increase in spinal flexibility after chymopapain injection is dose dependent and determine the "optimal" dosage of chymopapain to increase spinal flexibility in a rabbit model. SUMMARY OF BACKGROUND DATA: Spinal instability after chymopapain injection may result in severe back pain. However, this undesired mechanical effect in treating disc herniation may provide a safe minimally invasive approach for anterior spinal release in scoliosis correction. METHODS: A total of 138 lumbar intervertebral discs from 46 New Zealand white rabbits were randomly injected with chymopapain at 6.25, 12.5, 25, 50, 75, and 100 picokatals (pKats)/0.05 mL/disc. The rabbits were killed 1 week after the injection, and the lateral bending stiffness of the spinal segments without posterior elements was determined. RESULTS: The lateral bending spinal stiffness showed no significant change after injection of 6.25 and 12.5 pKats/0.05 mL/disc but reduced significantly following chymopapain injection of 25, 50, 75, and 100 pKats (all P < 0.05 by post hoc least significant difference tests). While the lateral bending stiffness was lowest at the 100-pKats dose, there were no significant differences between the four higher dosages. CONCLUSION: The reduction in the lateral bending spinal stiffness after chymopapain injection is dose dependent, and an optimal dosage for spinal release existed; doses greater than the optimal dosage did not result in further significant decrease in lateral bending spinal stiffness.

Three-dimensional dynamical measurement of upper limb support during paraplegic walking.

Functional electrical stimulation (FES) has been employed in paraplegic rehabilitation to resume their walking ability. However, there is less quantitative assessment method of FES walking efficiency and rehabilitation progress. This paper presents a new dynamical measurement of upper limb support force during paraplegic walking, which can be used to calculate the 3-D handle reaction vector (HRV). HRV may provide an assessment of FES-assisted efficiency. With a series of tests, the measurement accuracy, nonlinearity, and crosstalk of the designed system are testified. The force measurement error is found below 1.01%, while nonlinearity and crosstalk are less than 2.90%, and 3.19%, respectively. This means that the implemented walker system is reliable for the measurement of HRV during FES-assisted walking. A clinical trial is performed with a paraplegic subject. With the monitoring of FES-assisted walking, the downward component of HRV is found to decrease, implying the decreasing force generated from lower limb. The decrease slope in downward load curve can indirectly indicate the FES efficiency change during walking. The experiment and clinical trial results show that a 3-D dynamical measurement system is successfully accomplished to indirectly assess FES efficiency of lower limbs using quantitated forces applied by the upper limbs of paraplegic patients.

Glove punctures in orthopaedic surgery.

From February 2001 to May 2001, 792 latex gloves used in 100 operations from three orthopaedic sub-specialties (paediatrics, hand and spine) were tested for puncture by a water infusion test. Sixty-nine gloves from 45 operations were punctured, giving an overall glove perforation rate of 8.7% (69/792) and an operative perforation rate of 45% (45/100). The hand operations had the lowest operative perforation rate (19.4%) while the spine operations had the highest (63.6%). The glove perforation rate increased in bony procedures (60% versus 22.5%), in procedures with major instrumentation (66% versus 18%) and in more lengthy procedures. The thumbs and the left index finger had more punctures than other parts. In addition, the glove perforation rate for single gloving was 9.6% (53/552) while that for the inner glove of double gloving was 0.8% (1/120). Based on these findings, we would like to recommend double gloving and regular glove changing in these high-risk surgeries.

Tibiocalcaneal fusion after talectomy in arthrogrypotic patients.

The authors evaluated the results of five arthrogrypotic patients who had tibiocalcaneal fusion after previous talectomy. The average follow-up was 5 years (range 2-12 years) after fusion, and seven feet were available for evaluation in terms of function, pain, and radiologic changes. Using the American Orthopaedic Foot and Ankle Society clinical rating score, the five patients scored an average rating of fair after the arthrodesis compared with a rating of poor prior to the fusion. Four patients noted a subjective improvement with regard to their function and ankle pain after the fusion. Two patients still reported pain over the ankle but not enough to affect their activities. All the patients had residual leg length discrepancies ranging from 1 to 2 cm, but none of them required shoe lifts to correct the deformity. All the feet examined showed a plantigrade foot with an average tibia-floor angle of 91 degrees in the sagittal plane and an average hindfoot valgus of 2.75 degrees in the coronal plane. Radiographic examination showed that two patients already showed signs of mild degenerative arthritis over the knee and midtarsal joints that were not present before the tibiocalcaneal fusion. Tibiocalcaneal fusion may improve the function and the pain symptoms of arthrogrypotic patients, but it can lead to early degenerative arthritis of the adjacent joints.

Gene therapy for new bone formation using adeno-associated viral bone morphogenetic protein-2 vectors.

Previous reports have suggested that bone morphogenetic protein (BMP) gene therapy could be applied for in vivo bone regeneration. However, these studies were conducted either using immunodeficient animals because of immunogenicity of adenovirus vectors, or using ex vivo gene transfer technique, which is much more difficult to handle. Adeno-associated virus (AAV) is a replication-defective virus without any association with immunogenicity and human disease. This study was conducted to investigate whether orthotopic new bone formation could be induced by in vivo gene therapy using AAV-based BMP2 vectors. To test the feasibility of this approach, we constructed an AAV vector carrying human BMP2 gene. Mouse myoblast cells (C2C12) transduced with this vector could produce and secrete biologically active BMP2 protein and induce osteogenic activity, which was confirmed by ELISA and alkaline phosphatase activity assay. For in vivo study, AAV-BMP2 vectors were directly injected into the hindlimb muscle of immunocompetent Sprague-Dawley rats. Significant new bone under X-ray films could be detected as early as 3 weeks postinjection. The ossification tissue was further examined by histological and immunohistochemical analysis. This study is, to our knowledge, the first to establish the feasibility of AAV-based BMP2 gene therapy for endochondral ossification in immunocompetent animals.

Isokinetic and isometric lifting capacity of Chinese in relation to the physical demand of job.

The aim of the study was to formulate normative data for the lifting capacities of a normal Chinese population, in order to establish a basic foundation for further studies and to investigate the relationship between individual attributes including age, gender, height, weight, job physical demand and each type of lifting capacity. Isokinetic and isometric lifting strength at low, waist and shoulder assessment levels were measured using the LIDO Workset II based on a sample of 93 normal Chinese adults (63 men and 30 women) between the ages of 21-51. The 50th percentile score for adult Chinese female's lifting strength was 17.71% lower than the American female while the adult Chinese male's lifting strength was 14.94% lower than the American male. Lifting forces were higher in the 20-40 year age group. The isometric work mode had considerable impact on the lifting capacities, with shoulder level having the highest lifting capacities. The gender and body weight had a significant positive correlation to lifting capacity while job physical demand had a moderate correlation. Age and body heights were weakly correlated to lifting capacity.

Application of time-frequency analysis to somatosensory evoked potential for intraoperative spinal cord monitoring.

OBJECTIVE: To investigate the improvement in the reliability of intraoperative spinal cord monitoring by applying time-frequency analysis to somatosensory evoked potentials (SEP). METHODS: 34 patients undergoing scoliosis surgery were studied. SEP were recorded during different stages of scoliosis surgery. Averaged SEP signals were analysed intraoperatively by short time Fourier transform (STFT). The time-frequency characteristics of SEP were observed during surgery. The main peak in the time-frequency interpretation of SEP was measured in peak time, peak frequency, and peak power. The changes in these variables were compared with the changes in latency and amplitude during different surgical stages. RESULTS: During different surgical stages, changes in peak times and peak powers were found to correlate with the changes in latency and amplitude, respectively. Peak time showed more variability than latency (p < 0.01), while peak power showed less variability than amplitude (p < 0.01). The peak frequency of SEP appeared to be unchanged during surgery. SEP signals were found to have specific time-frequency characteristics, with the time-frequency distribution of the signals being located in a particular time-frequency space. CONCLUSIONS: Time-frequency analysis of SEP waveforms reveals stable and easily identifiable characteristics. Peak power is recommended as a more reliable monitoring parameter than amplitude, while peak time monitoring was not superior to latency measurement. Applying time-frequency analysis to SEP can improve the reliability of intraoperative spinal cord monitoring.

Controllable porosity hydroxyapatite ceramics as spine cage: fabrication and properties evaluation.

A procedure was designed to prepare porosity-graded hydroxyapatite (HA) ceramics simulating the bimodal structure of natural bone, which could be used to build a cage that would promote the reconstruction of the anterior column after vertebrectomy or corpectomy in tumor and trauma surgery. HA ceramics with controllable pore size distribution and porosity were developed by using chitosan and Poly(vinyl alcohol) (PVA) as the pore-forming agents. HA ceramics with worthwhile properties such as a wide range of volume porosity (10-50%) and pore size (nanometer to 400 microm) can be obtained from this method, which allows the fabrication of HA ceramics with desirable porous characteristics simulating the bimodal natural bone architecture expected to provide advantages for bony fusion in the intervertebral foramina. When coated with chitosan-gelatin network, the bending strength of the porous HA ceramics significantly improved. The polymer network coated porous HA have potential application in the construction of cages for spinal operations.

A comparison of deconvolution techniques for stress relaxation.

Stress relaxation (or equivalently creep) allows a large range of the relaxation (retardation) spectrum of materials to be examined, particularly at lower frequencies. However, higher frequency components of the relaxation curves (typically of the order of Hertz) are attenuated due to the finite time taken to strain the specimen. This higher frequency information can be recovered by deconvolution of the stress and strain during the loading period. This paper examines the use of three separate deconvolution techniques: numerical (Fourier) deconvolution, semi-analytical deconvolution using a theoretical form of the strain, and deconvolution by a linear approximation method. Both theoretical data (where the exact form of the relaxation function is known) and experimental data were used to assess the accuracy and applicability of the deconvolution methods. All of the deconvolution techniques produced a consistent improvement in the higher frequency data up to the frequencies of the order of Hertz, with the linear approximation method showing better resolution in high-frequency analysis of the theoretical data. When the different deconvolution techniques were applied to experimental data, similar results were found for all three deconvolution techniques. Deconvolution of the stress and strain during loading is a simple and practical method for the recovery of higher frequency data from stress-relaxation experiments.