Mineralized Collagen Matrix Research Articles

Differences between top-down and bottom-up approaches in mineralizing thick, partially demineralized collagen scaffolds

Biominerals exhibit complex hierarchical structures derived from bottom-up self-assembly mechanisms. Type I collagen serves as the building block for mineralized tissues such as bone and dentin. In the present study, 250–300μm thick, partially demineralized collagen scaffolds exhibiting a gradient of demineralization from the base to surface were mineralized using a classical top-down approach and a non-classical bottom-up approach. The top-down approach involved epitaxial growth over seed crystallites. The bottom-up approach utilized biomimetic analogs of matrix proteins to stabilize amorphous calcium phosphate nanoprecursors and template apatite nucleation and growth within the collagen matrix. Micro-computed tomography and transmission electron microscopy were employed to examine mineral uptake and apatite arrangement within the mineralized collagen matrix. The top-down approach could mineralize only the base of the partially demineralized scaffold, where remnant seed crystallites were abundant. Minimal mineralization was observed along the surface of the scaffold; extrafibrillar mineralization was predominantly observed. Conversely, the entire partially demineralized scaffold, including apatite-depleted collagen fibrils, was mineralized by the bottom-up approach, with evidence of both intrafibrillar and extrafibrillar mineralization. Understanding the different mechanisms involved in these two mineralization approaches is pivotal in adopting the optimum strategy for fabricating novel nanostructured materials in bioengineering research.

Cages Augmented With Mineralized Collagen and Platelet-Rich Plasma as an Osteoconductive/Inductive Combination for Interbody Fusion

After anterior cervical discectomy, fusion was radiologically, biomechanically, and histologically assessed in a sheep spine fusion model. To evaluate the efficacy of a platelet-rich plasma (PRP) application combined with a mineralized collagen matrix (MCM) as an alternative to autologous cancellous iliac crest bone grafts in a spine fusion model. PRP has the ability to stimulate bone and tissue healing. MCM is a recently developed osteoconductive material. Up to now, no comparative evaluation of PRP in combination with a MCM at the cervical spine has been performed in vivo. Twenty-four sheep (N = 8/group) underwent C3/4 discectomy and fusion: group 1, titanium cage filled with autologous cancellous iliac crest bone graft; group 2, titanium cage filled with MCM; and group 3, titanium cage filled with MCM and PRP. Radiographic evaluation was performed before surgery and after 1, 2, 4, 8, and 12 weeks, respectively. After 12 weeks, fusion sites were evaluated using functional radiographic views and quantitative computed tomographic scans to assess bone mineral density. Furthermore, histomorphologic and histomorphometrical analyses were performed to evaluate fusion. In comparison with the titanium cage group filled with autologous cancellous iliac crest bone grafts representing the control group, MCM-alone group showed a slightly lower fusion rate in the radiographic and the histomorphometrical analysis. The addition of PRP could not enhance this finding. There was no significant difference between MCM and MCM + PRP group in radiologic and histologic findings. The MCM alone is not able to replace autologous bone grafts. Early activation of the platelets by calcium, which is released from mineralized collagen, could be the reason for the insufficient osteoinductive effect of PRP. In consequence, the combined application of mineralized collagen and PRP had no significant osteoinductive effect in this model.

Layer by layer deposition of hydroxyapatite onto the collagen matrix

Layer by layer (LbL) deposition is a useful method for deposition of many inorganic (including metals, oxides and phosphates) and organic (including polymers and proteins) components on a large range of substrates. The LbL deposition of hydroxyapatite (HA) onto a collagen matrix involves HA synthesis on the collagen matrix starting from electrically charged precursors such as Ca 2+ and PO 4 3− at a proper pH to precipitate the desired calcium phosphate. The LbL deposition process was continuously monitored in order to study the amount of HA deposited in each layer. The deposition of the first layers of HA was concluded to be highly influenced by the collagen matrix. When the collagen matrix is crosslinked with glutaraldehyde, the matrix structure is not modified during the deposition, and the porosity will decrease with the number of layers until saturation is reached. Following pore saturation, HA will be only deposited onto the mineralized collagen matrix surface. The obtained composite materials were characterized by XRD, SEM, DTA-TG and FTIR.

Aging and the reduction in fracture toughness of human dentin

An evaluation of the crack growth resistance of human coronal dentin was performed on tissue obtained from patients between ages 18 and 83. Stable crack extension was achieved over clinically relevant lengths ( 0 ≤ a ≤ 1 mm ) under Mode I quasi-static loading and perpendicular to the nominal tubule direction. Results distinguished that human dentin exhibits an increase in crack growth resistance with extension (i.e. rising R -curve) and that there is a significant reduction in both the initiation (K o) and plateau (K p ) components of toughness with patient age. In the young dentin ( 18 ≤ age ≤ 35 ) there was a 25% increase in the crack growth resistance from the onset of extension (K o=1.34 MPa m 0.5) to the maximum or “plateau” toughness (K p=1.65 MPa m 0.5). In comparison, the crack growth resistance of the old dentin ( 55 ≤ age ) increased with extension by less than 10% from K o =1.08 MPa m 0.5 to K p =1.17 MPa m 0.5. In young dentin toughening was achieved by a combination of inelastic deformation of the mineralized collagen matrix and microcracking of the peritubular cuffs. These mechanisms facilitated further toughening via the development of unbroken ligaments of tissue and posterior crack-bridging. Microstructural changes with aging decreased the capacity for near-tip inelastic deformation and microcracking of the tubules, which in turn suppressed the formation of unbroken ligaments and the degree of extrinsic toughening.

Changes in subchondral bone mineral density and collagen matrix organization in growing horses

Objective The effects of growth and maturation on the mineral deposition and the collagen framework of equine subchondral bone (SCB) were studied. Materials and methods Osteochondral specimens (diameter 6 mm) from the left metacarpophalangeal joint of 5-( n = 8), 11-( n = 8) and 18-month-old ( n = 6) horses were investigated at two differently loaded sites ( Site 1 (S1): intermittent peak loading; Site 2 (S2): habitual loading). The SCB mineral density (BMD) was measured with peripheral quantitative computer tomography (pQCT), and the data were adjusted against the volume fraction (Vv) of the bone extracellular matrix (ECM). Polarised light microscopy (PLM) was used to analyze the Vv, the collagen fibril parallelism index and the orientation angle distribution in two fractions (1 mm/fraction) beneath the osteochondral junction of the SCB. PLM analysis was made along two randomly selected perpendicularly oriented vertical sections to measure the tissue anisotropy in the x-, y-, and z-directions. Results The BMD of SCB at S1 and S2 increased significantly during maturation. At the same time, the Vv of the ECM increased even more. This meant that the Vv-adjusted BMD decreased. There were no significant differences between sites. The basic collagen fibril framework of SCB seems to be established already at the age of 5 months. During maturation, the extracellular matrix underwent a decrease in collagen fibril parallelism but no changes in collagen orientation. The variation was negligible in the collagen network estimates in the two section planes. Conclusions Growth and maturation induce significant changes in the equine SCB. The BMD increase in SCB is primarily due to the growth of bone volume and not to any increase in mineral deposition. An increase in weight-bearing appears to greatly affect the BMD and the volume of the extracellular matrix. Growth and maturation induce a striking change in collagen fibril parallelism but not in fibril orientation. The structural anisotropy of the subchondral bone is significant along the vertical ( x– y) direction but not in the transversal ( z) direction.

Instrumented fusion of thoracolumbar fracture with type I mineralized collagen matrix combined with autogenous bone marrow as a bone graft substitute: a four-case report

In order to avoid the morbidity from autogenous bone harvesting, bone graft substitutes are being used more frequently in spinal surgery. There is indirect radiological evidence that bone graft substitutes are efficacious in humans. The purpose of this four-case study was to visually, manually, and histologically assess the quality of a fusion mass produced by a collagen hydroxyapatite scaffold impregnated with autologous bone marrow aspirate for posterolateral fusion. Four patients sustained an acute thoracolumbar fracture and were treated by short posterior segment fusion using the AO fixateur interne. Autologous bone marrow (iliac crest) impregnated hydroxyapatite-collagen scaffold was laid on the decorticated posterior elements. Routine implant removal was performed after a mean of 15.3 months (12–20). During this second surgery, fusion mass was assessed visually and manually. A bone biopsy was sent for histological analysis of all four cases. Fusion was confirmed in all four patients intraoperatively and sagittal stress testing confirmed mechanical adequacy of the fusion mass. Three out of the four (cases 2–4) had their implants removed between 12 and 15 months after the index surgery. All their histological cuts showed evidence of newly formed bone and presence of active membranous and/or enchondral ossification foci. The last patient (case 1) underwent implant removal at 20 months and his histological cuts showed mature bone, but no active ossification foci. This four-case report suggests that the fusion mass produced by a mineralized collagen matrix graft soaked in aspirated bone marrow is histologically and mechanically adequate in a thoracolumbar fracture model. A larger patient series and/or randomized controlled studies are warranted to confirm these initial results.

A preliminary comparative study of radiographic results using mineralized collagen and bone marrow aspirate versus autologous bone in the same patients undergoing posterior lumbar interbody fusion with instrumented posterolateral lumbar fusion

Background context Multiple bone graft substitutes for spinal fusion have been studied with varying results. Purpose The purpose of this study was to assess the effectiveness of a mineralized collagen matrix combined with bone marrow, versus autologous bone, in the same patients undergoing a posterior lumbar interbody fusion and an instrumented posterolateral lumbar fusion. Study design/setting A prospective, comparative study. Patient sample Patients indicated for one-level posterior lumbar interbody fusion and instrumented posterolateral lumbar fusion, serving as self-controls. Outcome measures Thin-cut computed tomographic scans with sagittal reconstruction and plain radiographs, including lateral flexion/extension views were performed and assessed at 12 and 24 months after surgery. Oswestry Disability Index and Visual Analog Scale questionnaires were completed by all patients preoperatively and at 12 and 24 months after surgery. Methods After informed consent and failure of nonoperative treatment, 25 consecutive patients requiring one-level instrumented posterolateral fusion combined with posterior interbody fusion were enrolled in the study. Mineralized collagen bone graft substitute combined with bone marrow aspirate was used on one side of the posterolateral fusion, with iliac crest autograft on the contralateral side. Results A fusion rate of 84% (21/25) was achieved for the autologous bone grafts and 80% (20/25) for the bone graft substitute. The interbody fusion rate was 92% (23/25). Mean Oswestry Disability Index (ODI) scores decreased 57.2% at 12 months and 55.6% at 24 months, compared with baseline. Conclusions Mineralized collagen bone graft substitute exhibited similar radiographic results compared with autograft in this model. Further trials incorporating bilateral fusion, as well as posterolateral fusion alone without interbody fusion are warranted to confirm the results of this study.

Osteogenesis induced by autologous bone marrow cells transplant in the pediatric skull

The ability of cranial bone to repair defects of continuity is limited and it is mostly dependent on the age of the patient. In infancy and in early pediatric age, the scarce thickness of the calvarial bones and the need for a harmonic development of the child's skull limit the application of most of the surgical procedures usually utilized in older patients. We tested the ability of mononucleated cells, derived from the patient's bone marrow and transplanted on the site of the cranial bone defect, to increase the rate of mineralization of the autologous osteogenesis to obtain the complete restoration of the skull continuity. Four children, aged 26, 28, 37, and 79 months, respectively, affected by a stabilized and persistent cranial bone defect of posttraumatic or postsurgical origin, were treated. A sandwich-shaped shell, made of extrused absorbable polylactic copolymers material, was used to hold in place a freeze-dried mineralized collagen matrix associated with a nonceramic hydroxyapatite scaffold, where autologous bone marrow mononucleated cells were inseminated. In all patients, a rapid autologous bone osteogenesis was observed with a clear dimensional reduction of the bone defect few months after the autologous bone marrow cells seeding. The preliminary results of this research suggest the use of autologous bone marrow cells to increase the autologous osteogenesis in early pediatric age in cases in which correction of skull bone defects is best realized with autologous bone.

6:00 184. Rabbit posterolateral fusion is achieved with recombinant human growth differentiation factor 5

BACKGROUND CONTEXT: Growth/differentiation factor-5 (GDF-5) is a member of a divergent subgroup of the bone morphogenetic protein (BMP) family. Members in this subgroup share 40–50% protein sequence homology with the BMP-2 and BMP-7 subgroups. Expression of GDF-5 is required for proper skeletal patterning and joint development in the vertebrate limb, and null mutations result in brachydactyly syndromes in both animal and man. Type I collagen and hydroxyapatite are the predominant components of bone. Fabricated from these osteoconductive components is a mineralized collagen matrix (further referred to as matrix) comparable in composition to natural bone. This matrix, combined with bone marrow aspirate as a source of osteogenic stimuli, is used clinically as a bone grafting material to fill bony voids. In previous animal studies, this matrix combined with rhGDF-5 demonstrated osteoinductive activity and promoted new bone formation in various bony defect and spine fusion models.

Alignment of Osteoblast-Like Cells and Cell-Produced Collagen Matrix Induced by Nanogrooves

Alignment of bone cells and collagen matrix is closely related to the anisotropic mechanical properties of bone. Intact scaffolds that promote osteoblast differentiation and mineralization in the preferred direction offer promise in the generation of biomimetic bone tissue. In this study, we examined the alignment of osteoblast-like cells and collagen fibers guided by nanogrooves. Nanoscale groove-ridge patterns (approximately 300 nm in periodicity, 60-70 nm in depth) on the surface of polystyrene (PS) were made by polarized Nd:YAG laser irradiation, at a wavelength of 266 nm. The influence of such "nanoscale features" on the orientation and alignment of cells and their mineralized collagen matrix was investigated, using rabbit mesenchymal stem cell (MSC)-derived osteoblast-like cells. The cells and actin stress fibers were aligned and elongated along the direction of the nanogrooves. In addition, the alignment of collagen matrix was also influenced by underlying nanogrooves. The results suggested that nanoscale fibrous cues in the longitudinal direction might contribute to the aligned formation of bone tissue. This may provide an effective approach for constructing biomimetic bone tissue.

Development of Controlled Heterogeneity on a Polymer-Ceramic Hydrogel Scaffold for Osteochondral Repair

Due to its intrinsically poor repair potential, injuries to articular cartilage do not heal and clinical intervention is required. Osteochondral grafts may improve healing while promoting integration with host tissue. We report here the development of an osteochondral graft based on a hybrid of a hyrogel and a polymer-bioactive glass composite (PLAGA-BG) microsphere scaffold. This novel osteochondral construct consists of three regions: gel-only, gel/composite interface, and a composite-only-region. The three phases differ in calcium phospate (Ca-P) or BG content. The objective of the current study is to investigate the effects of scaffold composition on chondrocyte response, and to evaluate the effects of co-culture on osteoblasts and chondrocyte growh and differentiation on the hybrid scaffold. The PLAGA-BG microsphere scaffold supported the growth of chondrocytes and initial results indicate that in the presence of BG, chondrocyte-mediated mineralization may be stimulated. Co-culture of osteoblasts and chondrocytes on the multi-phased scaffold with varied Ca-P content facilitated the formation of multiple matrix zones: a GAGrich chondrocyte region, an interfacial matrix rich in GAG+collagen, and a mineralized collagen matrix with osteoblasts. In summary, chondrocyte response has been optimized as a function of scaffold composition and the novel osteochondral graft has the potential to support the simultaneous formation of multiple types of tissue in vitro.

Lumbar spinal fusion with a mineralized collagen matrix and rhBMP-2 in a rabbit model.

A new mineralized collagen matrix combined with or without growth factor was used for the posterolateral spinal fusion in the rabbit lumbar spine. The availability of a new osteoconductive matrix with or without recombinant osteoinductive growth factors offers a possible alternative to the use of autogenous bone for grafting indications. This study evaluated the bone-forming activity of the biomimetic matrix: nano-hydroxyapatite/collagen/polylactic acid (nHAC/PLA) combined without or with recombinant human bone morphogenetic protein-2 (rhBMP-2) in a rabbit posterolateral spinal fusion. Many bone grafting materials such as titanium alloy, ceramics, and polymers were used to repair bony defects. However, each has specific disadvantaged. The permanent implantation still has possibility to be eroded in vivo, which is caused by late breakdown and abscess formation. The acidic outcome of polymer biodegradation was also negatively affected in the later-stage results of bone repair. It needed a promising material for an alternative to the use of autogenous bone for grafting indications. Sixty-four rabbits were randomly divided into four groups: autologous iliac crest bone group (ACB), nHAC/PLA composite group (nHAC/PLA), autologous iliac crest bone mixed with nHAC/PLA composite group (ACB + nHAC/PLA), nHAC/PLA composite with recombinant human BMP-2 group (nHAC/PLA + rhBMP-2). The lumbar intertransverse process fusions were assessed by manual palpation, radiographic, histologic, and mechanical strength, and scanning electronic microscopy (SEM) in a 10-week observation. Optimal formulations of the ACB + nHAC/PLA and nHAC/PLA + rhBMP-2 groups were shown to perform similar to ACB in both the fusion ratio and mechanical strength in the 6 and 10 weeks after surgery. From the microstructure analysis of the samples, there was no negative effect when the compound implanted this composite with autogenous iliac crest, and there was also new bone-like tissue formation implanted this composite without combined rhBMP-2 early at the second week after surgery. This study shows the effective results of nHAC/PLA in rabbit posterolateral spinal fusion combined with rhBMP-2. It is an alternative method to autograft by compounding this osteoconductive matrix with growth factors.

A recombinant bone growth factor and a mineralized collagen matrix (Healos/MP52) in threaded interbody fusion cages

James Maxwell, MD, Scottsdale, AZ, USA; Nicholas Theodore, MD, Phoenix, AZ, USA; Thomas Gilbert, MD, St. Louis Park, MN, USA; Bettina Willie, MS, Roy Bloebaum, PhD, Salt Lake City, UT, USA; Nathaniel R. Ordway, MS, Hansen A. Yuan, MD, Syracuse, NY, USA; Steven Griffith, PhD, Minneapolis, MN, USAIntroduction: Autograft has typically been used for interbody cage fusions of the spine with positive results. Yet, there is a need to provide alternatives to autogenous bone grafts. A potential osteopromotive growth factor is rhMP52 (also referred to as bone morphogenetic protein [BMP]-14, GDF-5 or CDMP-1) a divergent member of the BMP family. In previous animal studies, a mineralized collagen matrix (Healos Bone Graft Substitute) has been shown histologically to be replaced with mature bone when combined with autogenous bone marrow. The purpose of this study was to evaluate the combination of rhMP52 and Healos (Healos/MP52) for the promotion of spinal fusion in a baboon model using anterior lumbar threaded interbody cages. The effectiveness of Healos/MP52 to produce spinal fusion in and around interbody cages was compared with autogenous iliac crest bone graft.Materials and methods: In each of eight mature baboons (Papio cynocephalus), two noncontiguous spinal motion segments, L3–L4 and L5–L6, were implanted with single-threaded cages (Sulzer Spinetech, Minneapolis MN) that were randomly filled with either autograft or Healos/MP52 (Orquest, Mountainview, CA). Radiographs were obtained immediately after surgery and at 3-month intervals until animals were killed at either 3 months (n=4) or 6 months (n=4). After removing the spines en bloc, quantitative computed tomography was performed in a blinded manner within each cage chamber on mid-disc axial images. Individual motion segments were tested for biomechanical stability (ie, stiffness and range of motion) nondestructively in axial compression (300 N), flexion-extension (+4.5 Nm), lateral bending (+3 Nm) and torsion (+4.5 Nm). Histologic evaluation included mid-sagittal undecalcified sectioning, microradiography, backscattered electron microscopy, histomorphometry, light microscopy and pathoanatomic evaluation performed by blinded, independent examiners. Fusion was evaluated by a consensus of independent reviewers on lateral radiographs and histologic consideration with a definition of bridging trabecular bone from one end plate to the other through the holes of the cage.Results: There were no neurologic injuries or complications associated with surgery. No systemic effects were detected at necropsy. There was no evidence of bony compromise within the spinal canal or of delayed neurologic dysfunction. There were no instances of hardware failure. In six of eight animals (75%), a preponderance of observers judged the Healos/MP52 level to exhibit a more robust bone growth response than the autograft level. Lateral radiographs of cages filled with Healos/MP52 showed evidence of bone growth and anterior bony bridging outside of the implant that often became fully consolidated by 6 months. Radiographic fusion success for Healos/MP52 was 33.3% and 58.3% at 3 and 6 months, respectively. In contrast, the autograft-filled cages demonstrated 25% fusion success at both time points. A time-dependent difference in computed tomography (CT) densitometry was demonstrated in the Healos/MP52 group: 386.0 + 121.8 HU versus 568.6 + 191.6 HU, for 3 and 6 months, respectively (p=.1958), but not in the autograft groups: 622.5 + 81.0 HU versus 719.5 + 224.4 HU (p=.1347). Between group comparison of CT densitometry at each time point was significantly different at 3 months (p=.0011) but not at 6 months (p=.0963). No statistical differences were detected in stiffness and range of motion between the autograft group versus the Healos/MP52 group for any of the motions tested at either follow-up point (p>.05). There was an apparent, although not statistically different, increase in stiffness with a concomitant decrease in the range of motion, when these groups were compared with intact, nonoperative motion segments. Histologic specimens revealed endochondral bone formation within the cages and periosteal bone formation anterior to the cages. The fraction amount of trabecular bone within cages filled with Healos/MP52 was 17.3% + 5.8% at 3 months and increased to 25.1% + 9.5% in the 6-month group (p=.024), indicating progressive growth of bone.Conclusion: In this animal model of interbody fusion, a recombinant human bioactive growth factor (rhMP52) combined with a mineralized collagen matrix (Healos) produced bony fusion in and around threaded cages and in a manner superior to autograft. No pathology or aberrant bone formation was observed. Clinical evaluation of this promising combination of materials is warranted.

Spinal fusion with recombinant human growth and differentiation factor-5 combined with a mineralized collagen matrix.

The availability of recombinant osteoinductive growth factors and new osteoconductive matrices offers an alternative to the use of autogenous bone (autograft) for grafting indications. This study evaluates the bone-forming activity of a mineralized collagen matrix combined with recombinant human growth and differentiation factor-5 in a rabbit posterolateral spinal fusion model. The activity of three distinct matrix-growth factor formulations is assessed by radiographic, histologic, and mechanical strength methods. Results show that the radiographic density, histologic quality, and mechanical strength of fusion at 12 weeks post-treatment rank consistently within the treatment groups. Optimal formulations are shown to perform similar to autograft in both the rate and strength of fusion. Fusion rates as high as 80% are observed within specific matrix/growth factor formulations. The average biomechanical strength of treated motion segments in the most efficacious formulation is 82% higher than that obtained with autograft, although this difference is not statistically significant. The fusion mass formed in response to matrix/growth factor formulations is composed of normal trabecular bone with a thin outer cortical plate and modest hematopoietic bone marrow. These results demonstrate that the combination of a mineralized collagen matrix with recombinant human growth and differentiation factor-5 maximizes the inherent conductive and inductive properties of each component, respectively, to provide an effective alternative to autograft for bone grafting procedures.

Bone formation by transplanted human osteoblasts cultured within collagen sponge with dexamethasone in vitro.

To apply osteoblasts to bone reconstruction, we proved that transplanted osteoblasts possessed the differentiated osteoblastic function and formed bonelike tissue in vivo after transplantation. First, we confirmed that dexamethasone (Dex) promoted the expression of osteoblastic phenotype in human osteoblast culture using reverse-transcription-polymerase chain reaction (RT-PCR). These osteoblasts were cultured for 10 days within collagen sponge, which consists of denatured type I collagen, in the presence or absence of 10(-7) M Dex. The osteoblasts along with collagen sponge were transplanted into the trapezius muscles of 8-week-old severe combined immunodeficiency (SCID) mice, and the transplants were harvested at 2, 4, 6, and 8 weeks. At 2 weeks, Dex-treated osteoblasts formed bonelike tissue, the quantity of which increased in a time-dependent manner to 8 weeks. This bonelike tissue was composed of mineralized collagen matrix newly synthesized by the transplanted osteoblasts. This mineralized matrix was separated from the osteoblasts by nonmineralized matrixlike osteoid. Furthermore, many osteocytic cells were observed in this mineralized matrix. A high expression of alkaline phosphatase (ALPase) and osteocalcin was detected in the transplanted cells surrounding the bonelike tissue. In situ hybridization for human-specific alu sequence indicated that newly formed bone was of donor origin. The transplants of nontreated cells failed to form bonelike tissue. The transplants of collagen sponge alone formed no bonelike tissue. These studies indicate that Dex-treated human osteoblasts possess the differentiated osteoblastic function and are able to form bone tissue in vivo. These new findings are of use in facilitating the application of osteoblasts to bone reconstruction.

Inductive activity of recombinant human growth and differentiation factor-5

Growth and differentiation factor-5 (GDF-5) is a divergent member of the transforming growth factor-beta/bone morphogenetic protein (BMP) superfamily that is required for proper skeletal patterning and development in the vertebrate limb. Based on the homology of GDF-5 with other bone-inducing BMP family members, the inductive activity of a recombinant form of human GDF-5 (rhGDF-5) was evaluated in a series of in vitro assays and in vivo bone-formation models. The in vitro response to rhGDF-5 resulted in the formation of chondrogenic nodules in fetal rat calvarial cells cultured in the context of collagen or collagen/hyaluronate extracellular matrices. Matrices loaded with rhGDF-5 induced ectopic cartilaginous and osseous tissue when implanted in subcutaneous or intramuscular sites. In non-human primate long-bone-defect and spinal-fusion models, rhGDF-5 combined with a mineralized collagen matrix induced bone formation in a manner equivalent to autogenous bone. These results highlight the unique potential of rhGDF-5 in a wide variety of orthopaedic applications.

Inductive activity of recombinant human growth and differentiation factor-5

Growth and differentiation factor-5 (GDF-5) is a divergent member of the transforming growth factor-β/bone morphogenetic protein (BMP) superfamily that is required for proper skeletal patterning and development in the vertebrate limb. Based on the homology of GDF-5 with other bone-inducing BMP family members, the inductive activity of a recombinant form of human GDF-5 (rhGDF-5) was evaluated in a series of in vitro assays and in vivo bone-formation models. The in vitro response to rhGDF-5 resulted in the formation of chondrogenic nodules in fetal rat calvarial cells cultured in the context of collagen or collagen/hyaluronate extracellular matrices. Matrices loaded with rhGDF-5 induced ectopic cartilaginous and osseous tissue when implanted in subcutaneous or intramuscular sites. In non-human primate long-bone-defect and spinal-fusion models, rhGDF-5 combined with a mineralized collagen matrix induced bone formation in a manner equivalent to autogenous bone. These results highlight the unique potential of rhGDF-5 in a wide variety of orthopaedic applications.

Shape and orientation of osteoblast-like cells (Saos-2) are influenced by collagen fibers in xenogenic bone biomaterial.

The surface topography of a substratum has been shown to influence the growth and morphology of cells in culture. In this study, human osteoblast-like cells (Saos-2) were cultured on two types of xenogenic biomaterials obtained from bovine bone. Both biomaterials were similar in architectural organization and surface topography, but they differed in matrix components. The first one was characterized by preservation of the mineralized collagen matrix, and the second by complete deproteinization which only preserved the mineral phase. Cells cultured at the surface of both biomaterials were observed using scanning electron microscopy. The beta 1-integrin subunit, known to bind cell and collagen, is the major integrin of the osteoblast. It was localized using immunogold in transmission electron microscopy. At the surface of the collagen-containing matrix, cells exhibited an elongated shape and oriented axis parallel to the underlying collagen bundles. The beta 1-integrin subunit was localized at the outer surface of cells, in close association with collagen and at the contact points between cells and biomaterials. In contrast, at the surface of the single mineral matrix, cells were round shaped with random disposition. Gold particles were found around the cells with no specific relation to the biomaterial. These results strongly suggest that the chemical nature of the surface of a bone biomaterial directly influences adhesion process, shape, and spatial organization of cultured osteoblastic cells.

A new mineralized collagen matrix for bone regeneration and growth factor delivery

A new mineralized collagen matrix for bone regeneration and growth factor delivery

Characterization of a 5-Fluorouracil-Enriched Osteoprogenitor Population of the Murine Bone Marrow

AbstractIn the presence of β-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 × 10s cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 × 104 cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 × 104or 1 of 5 × 103 plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.