Moyo C. Kruyt

Tissue engineering of bone

Bone is a dynamic, highly vascularized tissue with the unique capacity to heal and to remodel depending on line of stress (Buckwalter et al, 1995ab). It exhibits the unlikely combination of high compressive strength and tensile strength due to the composite of calcium phosphate salts (hydroxyapatite) and collagen, respectively (Yaszemski et al, 1996a). It is difficult to find materials to mimic such a complex system when filling bone defects. However, current research capitalizes on the dynamic properties of bone by providing a biodegradable scaffold to guide healing.

Viable osteogenic cells are obligatory for tissue-engineered ectopic bone formation in goats

In this study we investigated the bone-forming capacity of tissue-engineered (TE) constructs implanted ectopically in goats. As cell survival is questionable in large animal models, we investigated the significance of vitality, and thus whether living cells instead of only the potentially osteoinductive extracellular matrix are required to achieve bone formation. Vital TE constructs of porous hydroxyapatite (HA) covered with differentiated bone marrow stromal cells (BMSCs) within an extracellular matrix (ECM) were compared with identical constructs that were devitalized before implantation. The devitalized implants did contain the potentially osteoinductive ECM. Furthermore, we evaluated HA impregnated with fresh bone marrow and HA only. Two different types of HA granules with a volume of approximately 40 microm were investigated: HA70/800, a microporous HA with 70% interconnected macroporosity and an average pore size of 800 microm, and HA60/400, a smooth HA with 60% interconnected macropores and an average size of 400 microm. Two granules of each type were combined and then treated as a single unit for cell seeding, implantation, and histology. The tissue-engineered samples were obtained by seeding culture-expanded goat BMSCs on the HA and subsequently culturing these constructs for 6 days to allow cell differentiation and ECM formation. To devitalize, TE constructs were frozen in liquid nitrogen according to a validated protocol. Fresh bone marrow impregnation was performed perioperatively (4 mL per implant unit). All study groups were implanted in bilateral paraspinal muscles. Fluorochromes were administered at three time points to monitor bone mineralization. After 12 weeks the units were explanted and analyzed by histology of nondecalcified sections. Bone formation was present in all vital tissue-engineered implants. None of the other groups showed any bone formation. Histomorphometry indicated that microporous HA70/800 yielded more bone than did HA60/400. Within the newly formed bone, the fluorescent labels showed that mineralization had occurred before 5 weeks of implantation and was directed from the HA surface toward the center of the pores. In conclusion, tissue-engineered bone formation in goats can be achieved only with viable constructs of an appropriate scaffold and sufficient BMSCs.

Comparative in vivo study of six hydroxyapatite-based bone graft substitutes

Improvement of synthetic bone graft substitutes as suitable alternatives to a patients own bone graft remains a challenge in biomaterials research. Our goal was to answer the question of whether improved osteoinductivity of a material would also translate to better bone‐healing orthotopically. Three porous biphasic calcium phosphate (BCP) ceramics (BCPA, BCPB, and BCPC), consisting of hydroxyapatite and β‐tricalcium phosphate, a porous biphasic calcium phosphate ceramic reinforced with a bioresorbable polylactic acid to improve its mechanical properties (BCPC+), a pure hydroxyapatite ceramic (HA), and a carbonated apatite ceramic (CA) were implanted intramuscularly and orthotopically by using a transverse process model in 11 goats for 12 weeks. BCPA and BCPB had similar chemical composition but differed in their microstructure. BCPB was not osteoinductive at all, but BCPA induced ectopic bone formation in 9 of 11 animals. Orthotopically, BCPA performed better than BCPB in both the amount and rate of bone formation. BCPC, similar to BCPA structurally and physicochemically, showed comparable results ectopically and orthotopically. Addition of resorbable polymer to BCPC made the material less osteoinductive (4 of 11 animals) and delayed bone formation orthotopically. Neither HA nor CA were osteoinductive, and their orthotopic performance was inferior to the osteoinductive ceramics. The results of the present study showed that material‐derived osteoinduction significantly enhanced bone healing orthotopically, and that this material property appeared more sensitive for predicting orthotopic performance than physicochemical and structural characteristics.

Genes for hereditary sensory and autonomic neuropathies: a genotype–phenotype correlation

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders characterized by axonal atrophy and degeneration, exclusively or predominantly affecting the sensory and autonomic neurons. So far, disease-associated mutations have been identified in seven genes: two genes for autosomal dominant (SPTLC1 and RAB7) and five genes for autosomal recessive forms of HSAN (WNK1/HSN2, NTRK1, NGFB, CCT5 and IKBKAP). We performed a systematic mutation screening of the coding sequences of six of these genes on a cohort of 100 familial and isolated patients diagnosed with HSAN. In addition, we screened the functional candidate gene NGFR (p75/NTR) encoding the nerve growth factor receptor. We identified disease-causing mutations in SPTLC1, RAB7, WNK1/HSN2 and NTRK1 in 19 patients, of which three mutations have not previously been reported. The phenotypes associated with mutations in NTRK1 and WNK1/HSN2 typically consisted of congenital insensitivity to pain and anhidrosis, and early-onset ulcero-mutilating sensory neuropathy, respectively. RAB7 mutations were only found in patients with a Charcot-Marie-Tooth type 2B (CMT2B) phenotype, an axonal sensory-motor neuropathy with pronounced ulcero-mutilations. In SPTLC1, we detected a novel mutation (S331F) corresponding to a previously unknown severe and early-onset HSAN phenotype. No mutations were found in NGFB, CCT5 and NGFR. Overall disease-associated mutations were found in 19% of the studied patient group, suggesting that additional genes are associated with HSAN. Our genotype–phenotype correlation study broadens the spectrum of HSAN and provides additional insights for molecular and clinical diagnosis.

Plate fixation versus intramedullary fixation for displaced mid-shaft clavicle fractures: a systematic review.

PurposeThe optimal surgical approach for displaced midshaft clavicle fracture remains controversial. The objective of this systematic review is to compare functional outcome and complications after plate fixation and intramedullary fixation for displaced midshaft clavicle fractures.MethodsA computer aided search of PUBMED and Embase was carried out on January 11th 2011. Every study that was published in the English, German, French or Dutch language was considered for inclusion. A total of four studies could be included of which two compared intramedullary fixation versus plate fixation, and two compared intramedullary fixation and plate fixation versus conservative treatment for displaced midshaft clavicle fractures. Studies that compared plate fixation with intramedullary fixation in patients with fresh unilateral displaced midshaft clavicle fractures were included. Dislocation or displacement had to be mentioned in the inclusion criteria of the study for inclusion in this review. The modified version of the Cochrane Bone, Joint and Muscle Trauma Group’s former quality assessment tool was used. Furthermore, the studies included were scored according to the GRADE approach to assess the quality. The chosen studies were summarised in a data-extraction form. Because of the different study designs and characteristics data were summarised separately for each study.ConclusionsHigh quality evidence from one study and low quality evidence from three studies showed no difference in functional outcome or complications after plate fixation or intramedullary fixation for displaced midshaft clavicle fractures.

Optimization of bone tissue engineering in goats: A peroperative seeding method using cryopreserved cells and localized bone formation in calcium phosphate scaffolds

Background. Bone tissue engineering by combining cultured bone marrow stromal cells with a porous scaffold is a promising alternative for the autologous bone graft. Drawbacks of the technique include the delay necessary for cell culture and the complicated logistics. We investigated methods to bypass these drawbacks. Furthermore, we investigated the localization of bone formation inside the scaffold. Methods. Bone marrow stromal cells from seven goats were culture expanded and cryopreserved. One week before surgery, some of the cells were thawed, cultured, and seeded on porous calcium phosphate scaffolds. The constructs were cultured for another week until implantation. The remaining cryopreserved cells were thawed just before implantation and peroperatively resuspended in plasma before combining with the scaffold. Scaffolds impregnated with fresh bone marrow, devitalized cultured constructs, and empty scaffolds served as controls. All samples were implanted in the back muscles of the goats for 9 weeks. Results. Histologic examination showed minimal (<1%) bone in the empty and devitalized scaffolds, 4.2±5.1 bone area percent in the bone marrow samples, and significantly more bone in both the cultured and peroperatively seeded constructs (11.7±2.5 and 14.0±2.0%). The peripheral 350 &mgr;m of the implants contained significantly less bone. Conclusion. Peroperative preparation of osteogenic constructs with cryopreserved cells is feasible. These constructs yield substantially more bone than the scaffolds alone or scaffolds impregnated with fresh bone marrow. Bone deposition is much less on the scaffold periphery.

Bone tissue engineering and spinal fusion: the potential of hybrid constructs by combining osteoprogenitor cells and scaffolds

In this paper, we discuss the current knowledge and achievements on bone tissue engineering with regard to spinal fusion and highlight the technique that employs hybrid constructs of porous scaffolds with bone marrow stromal cells. These hybrid constructs potentially function in a way comparable to the present golden standard, the autologous bone graft, which comprises besides many other factors, a construct of an optimal biological scaffold with osteoprogenitor cells. However, little is known about the role of the cells in autologous grafts, and especially survival of these cells is questionable. Therefore, more research will be needed to establish a level of functioning of hybrid constructs to equal the autologous bone graft. Spinal fusion models are relevant because of the increasing demand for graft material related to this procedure. Furthermore, they offer a very challenging environment to further investigate the technique. Anterior and posterolateral animal models of spinal fusion are discussed together with recommendations on design and assessment of outcome parameters.

Analysis of the Dynamics of Bone Formation, Effect of Cell Seeding Density, and Potential of Allogeneic Cells in Cell-Based Bone Tissue Engineering in Goats

After decades of research, relatively little is known about the role of bone marrow stromal cells (BMSCs) for bone tissue engineering. Although homogeneous cell seeding is regarded optimal, cell survival in large constructs is unlikely, except for the very periphery. Also no minimal and optimal BMSC densities have been identified. An interesting development is the use of allogeneic BMSCs. These have not yet been compared directly to autologous BMSCs. Culture-expanded BMSCs of 10 Dutch milk goats were cryopreserved and peroperatively seeded on 7 mm cubic scaffolds of 65% porous biphasic calcium phosphate (BCP). A range of BMSC densities (per cm3 scaffold) were prepared of 8E2 (= 8 x 10(2)), 8E3, 8E4, 8E5, 8E6 (considered the standard), and 1.6E7. Each goat received a control without cells, the six densities, and an 8E6 allogeneic BMSCs construct intramuscularly. After 3, 5, and 7 weeks, fluorochrome markers were administrated. At 9 weeks, implants were retrieved. The BCP scaffolds appeared to be autoinductive as the controls (without BMSCs) showed some bone. Early bone formation (before 3 weeks) appeared only at the peripheral 2mm of the BMSC-seeded constructs; the later 5- and 9-week labels were found more centrally, suggesting bone migration to the center. There was a minimum of 8E4 and optimum of 8E6 BMSCs/cm3. Allogeneic cells yielded comparable new bone.

Application and limitations of chloromethyl-benzamidodialkylcarbocyanine for tracing cells used in bone Tissue engineering.

Bone tissue engineering has the potential to provide us with an autologous bone substitute. Despite extensive research to optimize the technique, little is known about the survival and function of the cells after implantation. To monitor the cells, in vivo labeling is the method of choice. In this study we investigated the use of the fluorescent membrane marker chloromethyl-benzamidodialkylcarbocyanine (CM-Dil) to label cells used in bone tissue engineering. When applying label concentrations up to 50 microM, cells could be labeled efficiently without negative effects on cell vitality, proliferation, or bone-forming capacity. Porous hydroxyapatite scaffolds were seeded with labeled cells, and up to 6 weeks after implantation in nude mice cells could be traced inside tissue-engineered bone. However, contrary to other reports concerning intramembranous labels, transfer of the label from labeled to unlabeled cells was detected. Transfer occurred both in vitro and in vivo between vital cells and between dead and living cells. To determine when in vivo label transfer happened, devitalized, labeled constructs were implanted for various time periods in nude mice. The presence of vital labeled cells inside these constructs, when evaluated at different implantation periods, indicated transfer of the label. Transfer occurred at 7 days postimplantation when 40 microM label was applied, whereas 10 microM labeled constructs showed transfer 10 days after implantation. These findings indicate that CM-Dil label is useful for in vivo tracing of cells for follow-up periods up to 10 days. This makes the label particularly useful for cell survival studies in tissue-engineered implants.

Orthopaedic management of Hurler’s disease after hematopoietic stem cell transplantation: a systematic review

ObjectiveThe introduction of hematopoietic stem cell transplantation (HSCT) has significantly improved the life-span of Hurler patients (mucopolysaccharidosis type I-H, MPS I-H). Yet, the musculoskeletal manifestations seem largely unresponsive to HSCT. In order to facilitate evidence based management, the aim of the current study was to give a systematic overview of the orthopaedic complications and motor functioning of Hurler’s patients after HSCT.MethodsA systematic review was conducted of the medical literature published from January 1981 to June 2010. Two reviewers independently assessed all eligible citations, as identified from the Pubmed and Embase databases. A pre-developed data extraction form was used to systematically collect information on the prevalence of radiological and clinical signs, and on the orthopaedic treatments and outcomes.ResultsA total of 32 studies, including 399 patient reports were identified. The most frequent musculoskeletal abnormalities were odontoid hypoplasia (72%), thoracolumbar kyphosis (81%), genu valgum (70%), hip dysplasia (90%) and carpal tunnel syndrome (63%), which were often treated surgically during the first decade of life. The overall complication rate of surgical interventions was 13.5%. Motor functioning was further hampered due to reduced joint mobility, hand dexterity, motor development and longitudinal growth.ConclusionStem cell transplantation does not halt the progression of a large range of disabling musculoskeletal abnormalities in Hurler’s disease. Although prospective data on the quantification, progression and treatment of these deformities were very limited, early surgical intervention is often advocated. Prospective data collection will be mandatory to achieve better evidence on the effect of treatment strategies.