Biomedical subjects

Haisheng Li

Publications and source records attributed to Haisheng Li.

At least 19 recordsLinked to original sources

PubMed2007

Experimental lumbar spine fusion with novel tantalum-coated carbon fiber implant.

Implants of carbon fiber composite have been widely used in orthopedic and spinal surgeries. However, studies using carbon fiber-reinforced cages demonstrate frequent appearance of fibrous layer interposed between the implant and the surrounding bone. The aim of the present study was to test the possibility of coating a biocompatible metal layer on top of the carbon fiber material, to improve its biological performance. Tantalum was chosen because of its bone compatibility, based on our previous studies. A novel spinal fusion cage was fabricated by applying a thin tantalum coating on the surface of carbon-carbon composite material through chemical vapor deposition. Mechanical and biological performance was tested in vitro and in vivo. Compress strength was found to be 4.9 kN (SD, 0.2). Fatigue test with 500,000 cycles was passed. In vitro radiological evaluation demonstrated good compatibility with X-ray and CT scan examinations. In vivo test employed eight pigs weighing 50 kg each. Instrumented lumbar spine fusion of L3/4 and L4/5 with these cages was performed on each pig. After 3 months, excellent bone integration property was demonstrated by direct contact of the cage with the host bone and newly formed bone. No inflammatory cells were found around the implant. Cages packed with two different graft materials (autograft and COLLOSS) achieved the same new bone formation. The present study proved that coating tantalum on top of the carbon-based implant is feasible, and good bone integration could be achieved.

Animals↗

PubMed2006

Effects of bone protein extract on human mesenchymal stem cells proliferation and differentiation.

Since its osteoinductive capacity has been established, demineralized bone matrix is considered a suitable alternative to bone autograft in the healing of osseous defects. The mechanisms of bone formation induction are still not fully understood. In this study we assessed the effects of a dispersion of bovine bone extract COLLOSS (BPE) with regard to proliferation and differentiation of a human mesenchymal stem cell line overexpressing human telomerase reverse transcriptase (hMSC-TERT). Proliferation rate was determined by (3)H-thymidine incorporation. The differentiation of hMSC-TERT cells to osteoblastic cells was assessed by means of measuring alkaline phosphatase activity and collagen synthesis in vitro. Both undifferentiated and osteoblast-differentiated hMSC-TERT cells were investigated for response to BPE. The metabolic responses to BPE were compared to unstimulated cells and cells stimulated with bovine collagen (COL). Undifferentiated hMSC-TERT cells responded to BPE with increased proliferation and decreased alkaline phosphatase activity. Osteoblastic differentiated hMSC-TERT cells had a diminished proliferative response and an increased alkaline phosphatase activity and collagen synthesis. Our study demonstrated significant metabolic effects of BPE on hMSC-TERT cells, which were highly dependant on the differentiated state of the cells.

Alkaline Phosphatase↗

PubMed2006

Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds.

Culture of osteogenic cells on a porous scaffold could offer a new solution to bone grafting using autologous human mesenchymal stem cells (hMSC) from the patient. We compared coralline hydroxyapatite scaffolds with pore sizes of 200 and 500 microm for expansion and differentiation of hMSCs. We cultivated the hMSC statically or in spinner flasks for 1, 7, 14 and 21 days and found that the 200-microm pore scaffolds exhibited a faster rate of osteogenic differentiation than did the 500-microm pore scaffolds as shown by an alkaline phosphatase activity assay and real-time reverse transcriptase polymerase chain reaction for 10 osteogenic markers. The 500-microm scaffolds had increased proliferation rates and accommodated a higher number of cells (shown by DNA content, scanning electron microscopy and fluorescence microscopy). Thus the porosity of a 3D microporous biomaterial may be used to steer hMSC in a particular direction. We found that dynamic spinner flask cultivation of hMSC/scaffold constructs resulted in increased proliferation, differentiation and distribution of cells in scaffolds. Therefore, spinner flask cultivation is an easy-to-use inexpensive system for cultivating hMSCs on small to intermediate size 3D scaffolds.

Biocompatible Materials↗

PubMed2006

Porous tantalum trabecular metal scaffolds in combination with a novel marrow processing technique to replace autograft.

INTRODUCTION: Interbody fusion requires a structural member to carry load while the autograft or osteoinductive agent stimulates bone formation. In the present study, we evaluated the potential use of extracted nucleated cells from bone marrow mixed in hyaluronic acid gel as an osteoinductive agent, in comparison to Collagraft loaded with nucleated cells or rhBMP-2 in the porous tantalum ring, in an anterior lumbar interbody fusion (ALIF) in pigs. METHODS: Four 3-month-old female Danish landrace pigs were employed in the current study. Bone marrow was collected by means of aspiration, from the medullary cavity of the proximal tibia. The nucleated cells were isolated with a Ficoll step gradient centrifugation. The cell adhered rate after 24 hours of cultivation and ALP activity in the osteogenic medium following 4 days of cultivation were measured. Cell numbers in the porous tantalum discs were assessed by CyQuant measurement, and fluorescent live/dead cell staining in the porous tantalum discs was performed after periods of 24 hours and 7 days of cultivation. The nucleated cells mixed in hyaluronic acid gel were cultivated on slides for 24 hours, 7 days and 21 days. The live/dead cell staining, ALP staining or osteocalcin staining, were performed. A porous tantalum ring was loaded with nucleated cells in hyaluronic acid gel or packed with Collagraft strips also with nucleated cells or rhBMP-2. Immediately after preparation, one of three implants was inserted into L2-3, L4-5 or L6-7 respectively. The pigs were killed 3 months postoperatively. The lumbar spine specimens were prepared for histological evaluation. RESULTS: The adhered rate, of the nucleated cells, was 2.26+/-0.56%. ALP activity was no different in the osteogenic culture compared to DMEM/10%FBS alone. Cell numbers and live/dead cells showed no difference in the porous tantalum discs. Histological appearance showed that nucleated cells mixed with hyaluronic acid gel, had more mature bone in the central hole of the porous tantalum ring, compared to Collagraft strips with nucleated cells or rhBMP-2. Bone volume fraction did not differ within the three porous tantalum rings; however, more marrow space in the central hole of the porous tantalum ring was present when nucleated cells mixed with hyaluronic acid gel (57.4%) compared to Collagraft strips with rhBMP-2 (29.7%). CONCLUSION: In the current study, we demonstrate that nucleated cells, which were isolated from bone marrow intraoperatively, could be used to replace autograft if nucleated cells mixed with hyaluronic acid gel or with Collagraft strips packed into the porous tantalum ring in the pig ALIF model.

Animals↗

PubMed2006

Ectopic bone induction by equine bone protein extract.

Demineralized bone matrix from horse has been reported to be osteoinductive. However, its performance was inferior to autogenous bone graft in terms of new bone formation. In the present experiment, an equine bone protein extract-COLLOSS E was investigated for its osteoinductivity in a rat model. At the mean time, carboxymethyl-cellulose (CMC) was tested as a potential carrier for the protein extract. 18 male Wistar rats (8 weeks) were employed in the experiment. Each rat was implanted randomly with the 2 of the following implants, one on each side of the abdominal muscle. 1) COLLOSS E lyophilisate. 2) PEEK ring holder. 3) 3% or 10% CMC .in gel or lyophilized form 4) COLLOSS E lyophilisate with 3% CMC, implanted as gel or in lyophilized form. 5) COLLOSS E suspension with 10% CMC, implanted as gel or in lyophilized form. The rats were followed up for 21 days. After termination, samples were subjected to macroscopic examination, plain radiograph, micro-CT and histological evaluations. The results showed that PEEK ring or CMC alone could not induce ectopic bone formation. COLLOSS E lyophilisate has a slightly higher (6 out of 7) positive bone formation rate over COLLOSS E/3% CMC (3 out of 5, both gel and lyophilized form), however, the difference is non-significant (p=0.36, Fisher's exact test). 10% CMC with COLLOSS E did not show ectopic bone formation when implanted as gel form (0/8), while 1 positive bone formation was found when implanted as the lyophilized form (1/4). Bone tissue volume ranged from 0 mm(3) to 23.1mm(3) for COLLOSS-E lyophilisate alone and 0 to 29.7mm(3) for COLLOSS E/3%CMC (gel or lyophilized form). We concluded that equine bone protein extract has the ability to induce ectopic bone formation in the rat model. CMC could be a potential carrier, however, further studies are needed to verify the proportion and efficacy.

Animals↗

PubMed2006

Molecular mechanism of osteochondroprogenitor fate determination during bone formation.

Osteoblasts and chondrocytes, which derive from a common mesenchymal precursor (osteochondroprogenitor), are involved in bone formation and remodeling in vivo. Determination of osteochondroprogenitor fate is under the control of complex hormonal and local factors converging onto a series of temporospatial dependent transcription regulators. Sox9, together with L-Sox5 and Sox6, of the Sox family is required for chondrogenic differentiation commitment, while Runx2/Cbfa 1, a member of runt family and Osterix/Osx, a novel zinc finger-containing transcription factor play a pivotal role in osteoblast differentiation decision and hypertrophic chondrocyte maturation. Recent in vitro and in vivo evidence suggests beta-catenin, a transcriptional activator in the canonical Wnt pathway, can act as a determinant factor for controlling chondrocyte and osteoblast differentiation. Here we focus on several intensively studied transcription factors and Wnt/beta-catenin signal molecules to illustrate the regulatory mechanism in directing commitment between osteoblast and chondrocyte, which will eventually allow us to properly manipulate the mesenchymal progenitor cell differentiation on bone and regeneration of cartilage tissue engineering.

Animals↗

PubMed2006

Optimizing viral and non-viral gene transfer methods for genetic modification of porcine mesenchymal stem cells.

INTRODUCTION: Mesenchymal stem cells (MSCs) provide an excellent source of pluripotent progenitor cells for tissue-engineering applications due to their proliferation capacity and differentiation potential. Genetic modification of MSCs with genes encoding tissue-specific growth factors and cytokines can induce and maintain lineage-specific differentiation. Due to anatomical and physiological similarities to humans, porcine research models have been proven valuable for the preclinical testing of tissue engineering protocols in large animals. The aim of this study was to evaluate optimized viral and non-viral ex vivo gene delivery systems with respect to gene transfer efficiency, maintenance of transgene expression, and safety issues using primary porcine MSCs as target cells. MATERIALS AND METHODS: MSCs were purified from bone marrow aspirates from the proximal tibiae of four 3-month-old Danish landrace pigs by Ficoll step gradient separation and polystyrene adherence technique. Vectors expressing enhanced green fluorescent protein (eGFP) and human bone morphogenetic protein-2 (BMP-2) were transferred to the cells by different non-viral methods and by use of recombinant adeno-associated virus (rAAV)-mediated and retroviral gene delivery. Each method for gene delivery was optimized. Gene transfer efficiency was compared on the basis of eGFP expression as assessed by fluorescence microscopy and fluorescence-activated flow cytometry. BMP-2 gene expression and osteogenic differentiation were evaluated by realtime quantitative RT-PCR and histochemical detection of alkaline phosphatase activity, respectively. RESULTS: Non-viral gene delivery methods resulted in transient eGFP expression by less than 2% of the cells. Using high titer rAAV-based vector up to 90% of the cells were transiently transduced. The efficiency of rAAV-mediated gene delivery was proportional to the rAAV vector titer applied. Retroviral gene delivery resulted in long-term transgene expression of porcine MSCs. A 26-fold increase in percentage of eGFP expressing cells (1.7%+/-0.2% versus 44.1% +/-5.0%, mean +/-SD) and a 68-fold increase in mean fluorescence intensity (327.4+/-56.6 versus 4.8+/-1.3) was observed by centrifugation of retroviral particles onto the target cell layer. Porcine MSCs that were BMP-2 transduced by optimized retroviral gene delivery demonstrated a significant increase in BMP-2 gene expression and showed increased osteogenic differentiation. Retrovirally transduced porcine MSCs were furthermore tested free of replication-competent viruses. DISCUSSION: The non-viral gene transfer methods applied were significantly less efficient compared to the viral methods tested. However, due to advantages with respect to safety issues and ease of handling, improvement of non-viral gene delivery to primary MSCs deserves further attention. The high efficiency of rAAV-mediated gene delivery observed at high titers can be explained by the ability of rAAV vector to transduce nondividing cells and by its tropism towards porcine MSCs. rAAV-mediated gene delivery resulted in transient transgene expression due to lack of stable AAV genome integration. MLV-mediated retroviral gene delivery can be considered a safe method for long-term transgene expression by porcine MSCs, and is therefore particularly attractive for advanced tissue engineering strategies requiring extended transgene expression.

Animals↗

PubMed2006

Endothelin-1-induced femoral head epiphyseal artery constriction is enhanced by long-term corticosteroid treatment.

BACKGROUND: With regard to the pathogenesis of corticosteroid-associated femoral head osteonecrosis, only intravascular and extravascular factors have been discussed. Wall constriction of the lateral epiphyseal arteries, which are the main supplying arteries within the femoral head, has not previously been investigated. This study examined the effect of long-term corticosteroid treatment on vasoconstriction of the lateral epiphyseal arteries of the femoral head in a porcine model. METHODS: The immature female Danish Landrace pig was chosen as the model for our study because femoral head osteonecrosis can be induced in this animal and because the lateral epiphyseal arteries of the femoral heads are of the appropriate size for myographic investigation. Twenty-four female Danish Landrace pigs (two siblings from each of twelve litters for a total of twelve sibling pairs) were divided by randomly separating the sibling pairs into two groups, with a total of twelve pigs in each group. One group of twelve animals received a daily dose of 100 mg of methylprednisolone orally for three months. The other group of twelve pigs served as controls and received no corticosteroids. After the animals were killed with an intravenous injection of pentobarbital, the lateral epiphyseal arteries of the femoral head were isolated from the cancellous bone. These arteries were mounted as ring preparations on a small-vessel myograph for measurement of isometric force development. The effects on the resected vessels of the vasoactive agents endothelin-1, noradrenaline, bradykinin, substance P, and nitrous oxide were investigated. RESULTS: Vasoconstriction induced by increasing doses of endothelin-1 was increased after three months of methylprednisolone treatment compared with the vasoconstriction in vessels from control animals. Sensitivity to noradrenaline was not altered by this treatment. After submaximal precontraction by noradrenaline, vasorelaxation was elicited by substance P, nitrous oxide, and increasing doses of bradykinin without differences between the corticosteroid-treated and the control groups. CONCLUSIONS: Endothelin-1-induced vasoconstriction of the epiphyseal arteries of the femoral head was increased in this corticosteroid-treated pig model. Such vasoconstriction may lead to a reduction in femoral head blood flow and to local ischemia and thus may be a newly described factor in the multifactorial pathomechanism of osteonecrosis of the femoral head.

Animals↗

PubMed2005

Mucormycosis spondylodiscitis after lumbar disc puncture.

Vertebral osteomyelitis due to mucormycosis is a rare but fulminant and fatal disease. Only one case has been reported in literature, with postmortem diagnosis. The present paper reports a female case of mucormycosis spondylodiscitis and vertebral osteomyelitis after lumbar disc puncture and radio frequency nucleoplasty. She subsequently underwent two surgical debridements, continuous local irrigation and drainage, together with local and systemic Amphotericin B treatments. The infection was controlled 4 months after the second debridement; however, there was no improvement in the neurological function at the most recent follow-up, 16 months after the surgery. The experience of this patient, though a single case, supports early recognition, surgical debridement, systemic and local antifungal treatment, closed irrigation and drainage as the keys to successful treatment.

Antifungal Agents↗

PubMed2005

Pedicle screw fixation enhances anterior lumbar interbody fusion with porous tantalum cages: an experimental study in pigs.

STUDY DESIGN: A porous tantalum implant (Hedrocel, Implex Corp., Allendale, NJ), designed to assist interbody lumbar fusion, was tested biologically in an experimental model. A total of 11 female Danish landrace pigs received 3 levels of anterior lumbar interbody fusion at L2-L3, L4-L5, and L6-L7. Each level was randomly assigned one of the procedures: (1) implantation of PT-ring with pedicle screw fixation (PSF), (2) implantation of a porous tantalum ring (PT-ring) stabilized anteriorly with staples, or (3) implantation of carbon fiber cage (CF-cage) stabilized anteriorly with 2 staples. Each implant was filled with autogenous iliac crest bone graft. OBJECTIVES: To evaluate the effects of PSF on the incorporation of autologous bone in a porous tantalum interbody device and to compare healing in PT-rings to that in CF-cages of autologous bone. SUMMARY OF BACKGROUND DATA: Despite the promising results that early clinical trials have shown, interbody fusion cage technology is still under debate because of uncertainties that include indications for surgery, criteria for fusion, material, cage design, cage subsidence, and the effect of immediate stabilization. METHODS: Pigs were euthanized 6 months after surgery. Fusion segments were evaluated by plain radiography, conventional radiograph tomography, and histology. RESULTS: Fusion segments with PSF had significantly fewer radiolucencies than the other 2 levels (P = 0.002). Improved interface healing and fusion rate were observed in PT-rings when supplemented with PSF (P = 0.03). His tomorphometric results showed that the percentage of bone and bone marrow space in the center of a PT-ring was not significantly different from that of its adjacent vertebral body, but PSF increased bone marrow and decreased fibrous tissue formation in a tantalum cage. However, a CF-cage had higher bone volume and lower bone marrow space inside the cage compared with its adjacent vertebral body (P < 0.001). Fibrous tissue formation inside and around a CF-cage was more than that of a PT-ring (P < 0.05). CONCLUSION: Interbody fusion using a PT-ring cage packed with autologous bone achieved higher interface healing and more reliable fusion when fixated with supplementary pedicle screws than did fixated anteriorly with 2 staples. A lesser amount of bone graft was required, and bone remodeling was enhanced in the PT-ring when compared to the CF-cage.

Animals↗

PubMed2005

The influence of alendronate treatment and bone graft volume on posterior lateral spine fusion in a porcine model.

STUDY DESIGN: An experimental animal study with randomized, paired control design was conducted using a porcine model. OBJECTIVES: The aim of this study was to evaluate the influence of alendronate treatment and the significance of different amounts of bone graft on posterior lateral spine fusion. SUMMARY OF BACKGROUND DATA: Treatment with bisphosphonates inhibits osteoclast-induced bone resorption and increases bone quality and density. It has been widely used clinically for treatment of osteoporosis. Bisphosphonates have been reported to elongate the callus remodeling process during fracture healing. Bisphosphonate treatment may modify bone graft healing and the remodeling process in spine fusion. The bone resorption phase exists during the healing process. Extensive bone graft resorption could reduce the basis for new bone formation, which could be an important factor for failure of spine fusion. Furthermore, different amounts of initially applied bone graft may influence spine fusion rate and bone graft incorporation process. METHODS: Twenty-two pigs were included in the study. Eleven pigs in the treatment group received alendronate 10 mg/day p.o. for 3 months after surgery. The other 11 pigs received no bisphosphonate and served as control group. Posterior lateral fusion with the CD Horizon system was performed on the lumbar spine using different amounts of autograft (4 g on one side and 8 g on the other side) in all animals. The fusion was evaluated using radiograph, CT scan, and histomorphometry at 3 months after operation. RESULTS: There was no statistical difference in either fusion rate or fusion mass volume between the two groups. The fusion rate based on radiograph was 75% on the 8-g autograft side and 45% on the 4-g side (P < 0.05). The mean volume of fusion mass was 2.36 cm3 on the 4-g side and 3.29 cm3 on the 8-g side (P < 0.01). No difference was found in either trabecular bone volume or fusion rate between treatment and control groups using histologic evaluation.The treatment group showed a higher fibrous tissue volume(P < 0.05), higher proportion of woven bone structure(P < 0.001), and lower bone marrow volume (P = 0.088) in the fusion mass. Different amounts of bone graft did not change the tissue composition of the fusion mass. CONCLUSIONS: Alendronate treatment in this study decreased fusion mass remodeling without inhibiting fusion rate. Increased amounts of autologous bone graft could improve the fusion rate in this experimental spine fusion study.

Administration, Oral↗

PubMed2005

Experimental anterior lumbar interbody fusion with an osteoinductive bovine bone collagen extract.

STUDY DESIGN: A randomized experimental porcine anterior lumbar interbody fusion. OBJECTIVE: To determine the osteoinductive potential of a bovine bone derived collagen lyophilisate for anterior interbody spinal fusion in an animal model. SUMMARY OF BACKGROUND DATA: Complications associated with autologous bone graft harvesting are still major concerns when performing spinal fusion surgery. A new formulation of bovine bone collagen-protein extracts (Colloss) has been shown to be effective in animal studies as a bone graft enhancer. The present study further tested its osteogenic potential in the anterior spinal fusion cage in a controlled animal study. METHODS: Twelve normal Danish landrace pigs of 50 kg each were chosen as experimental animals. Lumbar spine interbody fusion of L3-L4, L4-L5 using titanium alloy cages was performed on each pig with pedicle screws instrumentation. Cages packed with either autograft or Colloss were randomly assigned to the two levels. Pigs were followed up to 3 months. After termination, the spine segments were evaluated by radiograph, micro-CT, and histomorphometry RESULTS: Ten pigs completed the study. Implant breakage, loosening, or spinal deformity was not observed after 12 weeks on radiograph examination. Micro-CT evaluations demonstrated that cages with Colloss had the same fusion rate (9 of 10) as those with autograft, further evaluation of bone volume (BV/TV), and trabecular thickness (Tb Th) also showed no different between them. Histologically, both autograft and Colloss levels had 80% (8 of 10) fusion rate. Histomorphometric results revealed that the only difference was the Colloss-filled cages had a higher cartilage tissue volume (P < 0.04). CONCLUSION: Colloss was effective as a bone graft substitute. It achieved the same fusion rate and the samepercentage of new bone formation as that of autologous iliac bone graft in this demanding porcine anterior spinal fusion model after 12 weeks.

Animals↗

PubMed2005

Cytokine profiles in conditioned media from cultured human intervertebral disc tissue. Implications of their effect on bone marrow stem cell metabolism.

BACKGROUND: Cytokines released from intervertebral discs cultured in vitro have not been profiled, and the effect of these cytokines on human bone marrow stem cells is yet to be studied. MATERIALS AND METHODS: Intervertebral discs from 14 patients who had undergone spinal fusion surgery were cultured separately in vitro. Conditioned media were collected after 48 and 96 h of culture in serum-free Minimum Essential Medium (MEM). Profiling of the cytokines was conducted using pooled media. Conditioned medium from each patient was also tested in human bone marrow stem cell culture, and incorporation of alkaline phosphatase and 3H-thymidine incorporation was evaluated. RESULTS: Of the 18 cytokines screened, 12 were found to be positive, but only eotaxin, IP-10, Rantes IL-6 and IL-8 seemed to be present at high levels. There was a close correlation between IL-6 and IL-8 levels in the medium (R = 0.90, p < 0.001). When the conditioned media were added to human bone marrow stem cell cultures, cellular proliferation was stimulated (p = 0.02), but alkaline phosphatase activity remained unchanged. Cellular proliferation correlated negatively with IL-6 levels (R = -0.44, p = 0.04). INTERPRETATION: Intervertebral discs secrete certain cytokines into the medium when cultured in vitro, and conditioned media from cultured intervertebral discs stimulate proliferation of bone marrow stem cells.

Adolescent↗

PubMed2004

Healing properties of allograft from alendronate-treated animal in lumbar spine interbody cage fusion.

This study investigated the healing potential of allograft from bisphosphonate-treated animals in anterior lumbar spine interbody fusion. Three levels of anterior lumbar interbody fusion with Brantigan cages were performed in two groups of five landrace pigs. Empty Brantigan cages or cages filled with either autograft or allograft were located randomly at different levels. The allograft materials for the treatment group were taken from the pigs that had been fed with alendronate, 10 mg daily for 3 months. The histological fusion rate was 2/5 in alendronate-treated allograft and 3/5 in non-treated allograft. The mean bone volume was 39% and 37.2% in alendronate-treated or non-treated allograft (NS), respectively. No statistical difference was found between the same grafted cage comparing two groups. The histological fusion rate was 7/10 in all autograft cage levels and 5/10 in combined allograft cage levels. No fusion was found at all in empty cage levels. With the numbers available, no statistically significant difference was found in histological fusion between autograft and allograft applications. There was a significant difference of mean bone volume between autograft (49.2%) and empty cage (27.5%) (P<0.01). In conclusion, this study did not demonstrate different healing properties of alendronate-treated and non-treated allograft for anterior lumbar interbody fusion in pigs.

Alendronate↗

PubMed2004

Anterior lumbar interbody fusion with carbon fiber cage loaded with bioceramics and platelet-rich plasma. An experimental study on pigs.

Platelet-rich plasma (PRP) is an autogenous source of growth factor and has been shown to enhance bone healing both in clinical and experimental studies. PRP in combination with porous hydroxyapatite has been shown to increase the bone ingrowth in a bone chamber rat model. The present study investigated whether the combination of beta tricalcium phosphate (beta-TCP) and PRP may enhance spinal fusion in a controlled animal study. Ten Danish Landrace pigs were used as a spinal fusion model. Immediately prior to the surgery, 55 ml blood was collected from each pig for processing PRP. Three-level anterior lumbar interbody fusion was performed with carbon fiber cages and staples on each pig. Autogenous bone graft, beta-TCP, and beta-TCP loaded with PRP were randomly assigned to each level. Pigs were killed at the end of the third month. Fusion was evaluated by radiographs, CT scanning, and histomorphometric analysis. All ten pigs survived the surgery. Platelet concentration increased 4.4-fold after processing. Radiograph examination showed 70% (7/10) fusion rate in the autograft level. All the levels with beta-TCP+PRP showed partial fusion, while beta-TCP alone levels had six partial fusions and four non-fusions ( P=0.08). CT evaluation of fusion rate demonstrated fusion in 50% (5/10) of the autograft levels. Only partial fusion was seen at beta-TCP levels and beta-TCP+PRP levels. Histomorphometric evaluation found no difference between beta-TCP and beta-TCP+PRP levels on new bone volume, remaining beta-TCP particles, and bone marrow and fibrous tissue volume, while the same parameters differ significantly when compared with autogenous bone graft levels. We concluded from our results in pigs that the PRP of the concentration we used did not improve the bone-forming capacity of beta-TCP biomaterial in anterior spine fusion. Both beta-TCP and beta-TCP+PRP had poorer radiological and histological outcomes than that of autograft after 3 months.

Animals↗

PubMed2004

Stimulation of porcine bone marrow stromal cells by hyaluronan, dexamethasone and rhBMP-2.

In the interest of optimizing osteogenesis in in vitro, the present study sought to determine how porcine bone marrow stromal cell (BMSc) would respond to different concentrations of hyaluronan (HY) and its different combinations with dexamethasone (Dex) and recombinant human bone morphogenic protein-2 (rhBMP-2). Cellular proliferation was determined by 3H-thymidine incorporation into DNA at both Days 2 and 7 when BMSc was cultivated with HY at concentrations of 0, 0.5, 1.0, 2.0 and 4.0 mg/ml. HY accelerated cellular proliferation when compared with cultures in the absence of HY at both Days 2 and 7. BMSc proliferation under the high HY concentration of 4 mg/ml was significantly higher than under the other, lower HY concentrations of 0.5, 1.0 and 2.0 mg/ml. When BMSc were cultivated under HY at concentrations of 0, 1.0 and 4.0 mg/ml and its 12 combinations with rhBMP-2 at concentrations of 0 and 10 ng/ml and Dex (+, -) at both Days 2 and 7, cellular responses were examined by 3H-thymidine incorporation into DNA, cellular alkaline phosphatase (ALP) activity, and pro-collagen type I C-terminal propeptide production. HY accelerated cellular proliferation irrespective of the presence of Dex and rhBMP-2. HY increased expression of ALP activity at Day 7, whereas had inhibitory effect at Day 2. HY and Dex showed an interaction on expression of ALP acitivity irrespective of the HY dose by Day 7. Collagen synthesis was inhibited by HY irrespective of the presence of other factors at both Days 2 and 7. When BMSc were cultivated with HY of 4.0 mg/ml alone, its combinations with Dex (+) and 10 ng/ml rhBMP-2, and with DMEM/FBS alone, expression of bone-related marker genes was evaluated by real-time reverse transcription-polymerase chain reaction (Real-time RT-PCR) analysis. Osteocalcin was up-regulated under both rhBMP-2 and HY-Dex-rhBMP-2 at Day 2, as also under 4 mg/ml HY, Dex, HY-Dex, Dex-rhBMP-2, and HY-Dex-rhBMP-2 by Day 7. Type 1alpha1 collagen was induced by rhBMP-2 on Day 2, and by Dex-rhBMP-2 on Day 7. Osteonectin and type X collagen was only marginally induced by HY at Day 2. Type 1alpha1 collagen and type X collagen were down-regulated in the presence of 4 mg/ml HY by Day 7. These results suggest that HY stimulates BMSc proliferation, osteocalcin gene expression, and a secretion of enzymes such as that of ALP activity in vitro. More importantly, HY can interact with Dex and rhBMP-2 to generate direct and specific cellular effects, which could be of major importance in bone tissue engineering.

Animals↗

PubMed2004

Effects of autogenous bone graft impaction and tricalcium phosphate on anterior interbody fusion in the porcine lumbar spine.

Background Impaction grafting can be achieved inside the spinal fusion cages, but its effect on bone graft incorporation and spinal fusion has not been studied. Animals and methods We investigated the effect of impaction grafting on the bone graft healing and fusion potential of beta-tricalcium phosphate (beta-TCP) inside the carbon fiber reinforced spinal fusion device (Brantigan cage) in 10 Danish landrace pigs. Lumbar spine interbody fusion of L2/3, L4/5 and L6/7 using carbon fiber cages was performed on each pig. Cages filled with either loosely-packed autologous iliac bone graft, rod-impacted autologous bone graft or beta-TCP were randomly distributed to the three fusion levels. Half of the animals were followed for 8 weeks, and the other half for 16 weeks. Results Radiographs and CT evaluations showed that autograft levels had significantly better results than beta-TCP levels (p<0.001 Fisher's Exact Test). However, the difference between impacted and loosely-packed levels was not significant. Histomorphometric analysis showed no difference between the loosely-packed and impacted cages with regard to bone volume, bone marrow volume, cartilage and fibrous tissue volume, while both of the autograft levels differed from the beta-TCP levels in all of the aforementioned parameters. Fluorochrome studies demonstrated that bone mineral apposition rate was significantly higher in the impacted cages than in the loosely-packed cages at 16 weeks. Interpretation Manual impaction of autologous bone graft into the carbon fiber cages resulted in a faster mineral apposition rate by 16 weeks. Bone ingrowth and spinal fusion were not influenced by impaction grafting.

Animals↗

PubMed2003

Inhibition of spinal fusion by use of a tissue ingrowth inhibitor.

"Spinal instrumentation without fusion" techniques, which do not interfere with spinal growth, have been used extensively in the treatment of progressive spinal scoliosis in very young children. Due to subperiosteal exposure, the process of spinal instrumentation may induce spontaneous bony fusion. Instrumentation and surgical techniques have been modified in order to prevent spontaneous posterior fusion from occurring in children. An absorbable ADCON-L gel has been shown to inhibit scar and epidural adhesions following spinal surgeries. However, little is known about its influence on spinal fusion. In the present study, a single-level intertransverse arthrodesis at L4-5 on both sides was performed on each of nine pigs. Each side was randomly designed to receive autogenous bone graft with or without ADCON-L gel. The animals were followed for 10 weeks postoperatively. A fusion rate of 78% (7/9) was obtained in the autograft treatment by plain X-ray and CT evaluation, while the autograft/ADCON-L treatment yielded a 0% (0/9) fusion rate ( p = 0.001). Histomorphometric evaluation revealed that the addition of ADCON-L gel to bone graft decreased bone and bone marrow formation and significantly increased fibrous tissue formation. No statistical difference between the two treatments was found in cartilage, bone surface density, osteoid surfaces or osteoclast-covered surfaces in any zone. We conclude that ADCON-L gel mixed into autogenous bone graft can delay or decrease bone formation at spinal arthrodesis sites, thus influencing the extent of spinal fusion. This accords with our hypothesis that the use of ADCON-L gel can prevent not only the occurrence of spontaneous fusion in very young scoliosis patients after instrumentation without fusion, but also re-ossification of a decompressed spinal canal.

Animals↗