Engineering of hyaline cartilage with a calcified zone using bone marrow stromal cells.

OBJECTIVE: In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage-substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones. DESIGN: BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days. RESULTS: This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone. CONCLUSION: This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies.

Processing and properties of Na-doped porous calcium polyphosphates - Mechanical properties and in vitro degradation characteristics.

Porous calcium polyphosphate (CPP) is being investigated for use as a biodegradable bone substitute and for repair of osteochondral defects. The necessary requirements for these applications, particularly in load-bearing sites, include sufficient strength to withstand functional forces prior to bone ingrowth and substitution of the initial porous CPP template with new bone and cartilage (for osteochondral implants) in a timely and efficacious manner. The present study explored the effects of Na+ doping and processing to form porous structures of both higher strength and faster degradation than previously reported for 'pure' (non-doped) CPP structures of similar geometry. Compressive and tensile strengths were determined before and after 30-day in vitro degradation (PBS, pH 7.1 at 37 °C) and degradation rates assessed. Scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid state nuclear magnetic resonance (31P SS NMR) were used to evaluate 'pure' and Na-doped CPP samples before and after degradation. The results indicated that the different processing protocols required to prepare samples of similar volume % porosity (a 2-step procedure with a Step-1 sintering temperatures equal to 575 °C being used with the Na-doped samples versus a 585 °C Step-1 treatment for 'pure' CPP) resulted in an approximate 1.5- to 2-fold increase in strength (tensile & compressive respectively) and 2-fold increase in degradation rate of Na-doped CPP compared with 'pure' CPP. This difference was attributed to the different Step-1 sintering temperatures used for sample processing.

AP-1 DNA binding activity regulates the cartilage tissue remodeling process following cyclic compression in vitro.

Generating bioengineered cartilage yields tissue with physical qualities inferior to that of native tissue. Application of cyclic compression (30 min, 1 kPa, 1 Hz) to cartilage cells (chondrocytes) seeded on calcium polyphosphate substrates significantly increases the accumulation of collagens and proteoglycans by 24 hours, thus improving the tissue generated. The mechanism for this increase is not fully known but seems to follow a remodeling pathway of sequential catabolic and anabolic changes. The initial catabolic event involves increased transcription of matrix metalloproteinase (MMP)-3 and MMP-13 two hours after the end of cyclic compression. As MMP-3 and MMP-13 promoters contain activating protein-1 (AP-1) DNA binding sites, we investigated the effect of inhibiting DNA binding through the use of modified decoy oligodeoxynucleotides (ODN). Mechanical stimulation in the presence of the ODN blocked AP-1 DNA binding as detected by electrophoretic mobility shift assay and prevented the increased transcription of MMP-3 and MMP-13. As well the increased accumulation of collagens and proteoglycans by 24 hours in mechanically stimulated samples was prevented. The data suggests that the mechano-induction of MMP-3 and MMP-13 may be regulated at the AP-1 DNA binding site and that upregulation of these metalloproteases is a necessary component of the matrix remodeling initiated by cyclic compression.

Formation of biphasic constructs containing cartilage with a calcified zone interface.

The zone of calcified cartilage is the mineralized region of articular cartilage that anchors the hyaline cartilage to the subchondral bone and serves to disperse mechanical forces across this interface. In an attempt to mimic this zonal organization, we have developed the methodology to form biphasic constructs composed of cartilaginous tissue anchored to the top surface of a bone substitute (porous calcium polyphosphate, CPP) with a calcified interface. To accomplish this, chondrocytes were selectively isolated from the deep zone of bovine articular cartilage, placed on top of the CPP substrate, and grown in the presence of beta-glycerophosphate (10 mM, beta-GP). By 8 weeks, cartilage tissue had formed with two zones: a calcified region adjacent to the CPP substrate and a hyaline-like zone above. Little or no mineralization occurred in the absence of beta-GP. The mineral that formed in vitro was identified as hydroxyapatite, similar in composition and crystal size to that found in vivo. The tissue stiffness was seven times greater, and the interfacial shear properties at the cartilage-CPP interface were at least two times greater in the presence of this mineralized zone within the in vitro-formed cartilage than in tissue lacking a mineral zone. In conclusion, developing a biphasic construct with a calcified zone at the tissue-biomaterial interface resulted in significantly better cartilage load-bearing (compressive) properties and interfacial shear strength, emphasizing the importance of the presence of a mineralized zone in bioengineered cartilage. Because failure due to shear occurred at the cartilage-CPP interface instead of the tidemark, as occurs with osteochondral tissue, further study is required to optimize this system so that it more closely mimics the native tissue.

Osteochondral defect repair using a novel tissue engineering approach: sheep model study.

Porous calcium polyphosphate (CPP) constructs of desired density were formed by sintering CPP powders. Articular cartilage was formed on these constructs in cell culture over an 8-week period with the resulting cartilage layer forming on the CPP surface and within the near surface pores thereby mechanically anchoring the cartilage to the CPP. The biphasic constructs so formed were implanted in sheep femoral condyle sites and left for short-term periods (3 to 4 months) or longer periods (9 months). Implant fixation within the condyle sites was achieved through bone ingrowth into the inferior CPP pores. The properties and characteristics of the as-in vitro-formed, short- and long-term implanted tissues were compared. The results indicated that such implants might be useful for repair of small subchondral defects.

Cyclic compressive mechanical stimulation induces sequential catabolic and anabolic gene changes in chondrocytes resulting in increased extracellular matrix accumulation.

Overcoming the limited ability of articular cartilage to self-repair may be possible through tissue engineering. However, bioengineered cartilage formed using current methods does not match the physical properties of native cartilage. In previous studies we demonstrated that mechanical stimulation improved cartilage tissue formation. This study examines the mechanisms by which this occurs. Application of uniaxial, cyclic compression (1 kPa, 1 Hz, 30 min) significantly increased matrix metalloprotease (MMP)-3 and MMP-13 gene expression at 2 h compared to unstimulated cells. These returned to constitutive levels by 6 h. Increased MMP-13 protein levels, both pro- and active forms, were detected at 6 h and these decreased by 24 h. This was associated with tissue degradation as more proteoglycans and collagen had been released into the culture media at 6 h when compared to the unstimulated cells. This catabolic change was followed by a significant increase in type II collagen and aggrecan gene expression at 12 h post-stimulation and increased synthesis and accumulation of these matrix molecules at 24 h. Mechanical stimulation activated the MAP kinase pathway as there was increased phosphorylation of ERK1/2 and JNK as well as increased AP-1 binding. Mechanical stimulation in the presence of the JNK inhibitor, SP600125, blocked AP-1 binding preventing the increased gene expression of MMP-3 and -13 at 2 h and type II collagen and aggrecan at 12 h as well as the increased matrix synthesis and accumulation. Given the sequence of changes, cyclic compressive loading appears to initiate a remodelling effect involving MAPK and AP-1 signalling resulting in improved in vitro formation of cartilage.

Substrate porosity enhances chondrocyte attachment, spreading, and cartilage tissue formation in vitro.

Tissue engineering is being explored as a new approach to treat damaged cartilage. As the biomaterial used may influence tissue formation, the effects of substrate geometry on chondrocyte behavior in vitro were examined. Articular chondrocytes were isolated and cultured on the surface of smooth, rough, porous-coated, and fully porous Ti-6Al-4V substrates. The percentage of chondrocytes that attached to each substrate at 24 h was determined. After 24 and 72 h, chondrocytes were visualized by scanning electron microscopy and cell areas were measured. Collagen and proteoglycan accumulation within the first 24 h was determined by incorporation with [3H]-proline and [35S]-SO4, respectively. Chondrocyte attachment as well as matrix accumulation was enhanced as substrate surface area increased. Cell areas on the fully porous substrate were over four times greater than on any other substrate by 72 h in culture. After 8 weeks in culture, a continuous layer of cartilaginous tissue formed only on the surface of the fully porous substrate. This suggests that fully porous Ti-6Al-4V substrates provide the conditions that favor cartilage tissue formation by influencing cell attachment and extent of cell spreading. Understanding how substrate porosity influences chondrocyte behavior may help identify methods to further enhance cartilage tissue formation in vitro.

The porous-surfaced electrode: a new concept in pacemaker lead design.

Three major problems which may be encountered with endocardial pacemaker electrodes are a lack of stable position, a chronic increase in stimulation threshold, and a diminishing magnitude of the sensed endocardial signal. These problems are particularly manifest in the atrium. Having previously shown that porous metal surfaces can support stable tissue ingrowth in both bloodstream and soft tissue environments, we set out todetermine the performance of porous-surfaced endocardial pacing electrodes in the atrial position. In two groups of six dogs each, J-shaped atrial leads with Elgiloy electrode tips (2.3 mm. in diameter, 2.3 mm. in length), having either conventional smooth surfaces (control) or porous surfaces (20 to 50 micron particle size) produced by powder metallurgy techniques, were positioned in the right atrial a-pendage. Stimulation thresholds and P-wave amplitude were repeatedly measured until the dogs were put to death 30 w-eks following implantation. The presence or absence of electrode fixation was observed and the atrial tissue reaction was examined grossly and by both light and scanning electron microscopy (SEM). The porous-surfaced electrodes demonstrated superior long-term stimulation thresholds which, at 30 weeks, averaged less then one third of those in the control group. In addition, the porous group showed a small but significant improvement in the amplitude of the sensed P wave. None of the smooth-surfaced electrodes showed fixation, and the tissue reaction consisted of a thick layer of granulation and fibrous tissue on the underlying endocardium, widely separating the electrode from the myocardium. In contrast, all of the porous-surfaced electrodes were fexed to the endocardium by fibrous tissue ingrowth into the surface pores. This tissue fixation of the electrode tip in close proximity to underlying myocytes explains their superior performance.

Modern metal processing for improved load-bearing surgical implants.

A review of modern methods for preparing metallic alloys that could be useful for the fabrication of load-bearing metallic biomaterials is presented. The use of rapid solidification processing and surface modification of metals by ion implantation or surface coatings and variations thereof is used currently for the formation of novel metallic alloys in other high-tech fields, notably the optoelectronics industry. Further studies to explore potential benefits for surgical implant fabrication through the application of these technologies is recommended.

The effect of sol-gel-formed calcium phosphate coatings on bone ingrowth and osteoconductivity of porous-surfaced Ti alloy implants.

Ti-6Al-4V implants formed with a sintered porous surface for implant fixation by bone ingrowth were prepared with or without the addition of a thin surface layer of calcium phosphate (Ca-P) formed using a sol-gel coating technique over the porous surface. The implants were placed transversely across the tibiae of 17 rabbits. Implanted sites were allowed to heal for 2 weeks, after which specimens were retrieved for morphometric assessment using backscattered scanning electron microscopy and quantitative image analysis. Bone formation along the porous-structured implant surface, was measured in relation to the medial and lateral cortices as an indication of implant surface osteoconductivity. The Absolute Contact Length measurements of endosteal bone growth along the porous-surfaced zone were greater with the Ca-P-coated implants compared to the non-Ca-P-coated implants. The Ca-P-coated implants also displayed a trend towards a significant increase in the area of bone ingrowth (Bone Ingrowth Fraction). Finally, there was significantly greater bone-to-implant contact within the sinter neck regions of the Ca-P-coated implants.

Porous calcium polyphosphate scaffolds for bone substitute applications in vivo studies.

Porous rods (6 mm in length and 4 mm in diameter) of calcium polyphosphate (CPP) made by gravity sintering of particles in the size ranges of 45-105, 105-150. and 150-250 microm and with initial volume percent porosity in the range of 35-45% were implanted in the distal femur of New Zealand white rabbits. In an initial experiment, four rabbits implanted with rods made from coarse particles (150-250 microm) were sacrificed at each of the following time points: 2 days, 2 weeks, 6 weeks and 12 weeks. In a subsequent experiment, 10 rabbits were implanted with rods made by sintering 45-105 microm particles and another 10 were made by using particles of 105-150 microm. These rabbits were sacrificed at 6 weeks (five rabbits) and 1 year (five rabbits). No adverse reaction was found histologically at any time point in either experiment. These experiments show that CPP macroporous rods can support bone ingrowth and that between 12 weeks and 1 year, the amount of bones formed is equivalent to the natural bone volume found at similar sites. The degradation of the CPP material is inversely proportional to the original particle size and is rapid initially (within the first 6 weeks) and slows down thereafter. In conclusion, this material seems to promote rapid bone ingrowth and can be tailored to degrade at a given rate in vivo to some degree through appropriate selection of the starting particle size.

Porous calcium polyphosphate scaffolds for bone substitute applications -- in vitro characterization.

Porous structures were formed by gravity sintering calcium polyphosphate (CPP) particles of either 106-150 or 150-250 microm size to form samples with 30-45 vol% porosity with pore sizes in the range of 100 microm (40-140 microm). Tensile strength of the samples assessed by diametral compression testing indicated relatively high values for porous ceramics with a maximum strength of 24.1 MPa for samples made using the finer particles (106-150 microm). X-ray diffraction studies of the sintered samples indicated the formation of beta-CPP from the starting amorphous powders. In vitro aging in 0.1 M tris-buffered solution (pH 7.4) or 0.05 M potassium hydrogen phthalate buffered solution (pH 4.0) at 37 degreesC for periods up to 30d indicated an initial rapid loss of strength and P elution by 1 d followed by a more gradual continuing strength and P loss resulting in strengths at 30d equal to about one-third the initial value. The observed structures, strengths and in vitro degradation characteristics of the porous CPP samples suggested their potential usefulness as bone substitute materials pending subsequent in vivo behaviour assessment.

Cell attachment of human gingival fibroblasts in vitro to porous-surfaced titanium alloy discs coated with collagen and platelet-derived growth factor.

The influence of biological coating, with or without the incorporation of growth factor, on the migration, attachment and orientation of human gingival fibroblasts in relation to porous-surfaced titanium alloy (Ti6AI4V) discs, was measured. Comparison was made between coating the discs with collagen and with collagen incorporating platelet-derived growth factor (PDGF); controls comprised porous-surfaced discs coated with agar or collagen containing bovine serum albumin (used as a carrier for the PDGF), uncoated porous-surfaced Ti6AI4V discs (with or without additional protein additives) exhibited significantly higher attachment indices (AI) and orientation indices (OI) compared with naked control discs (p less than 0.01); OI was also significantly higher than that of surface-demineralized root slices (p less than 0.001) on days 1, 2 and 3. Addition of PDGF to the collagen resulted in a further enhancement in OI on days 1 and 2 (p less than 0.01) over that shown by discs coated with collagen incorporating the bovine serum albumin vehicle. There was no cell attachment and consequently, no cell orientation, in relation to Ti alloy discs that had been coated with agar. These data suggest that attachment and orientation of cells following migration in relation to porous-surfaced Ti6AI4V discs can be modified by the application of biological molecules to the surface of the disc. This may have a useful application in clinical implantology.

Differences in osseointegration rate due to implant surface geometry can be explained by local tissue strains.

Experimental evidence indicates that the surface geometry of bone-interfacing implants influences the nature and rate of tissues formed around implants. In a previously reported animal model study, we showed that non-functional, press-fitted porous-surfaced implants placed in rabbit femoral condyle sites osseointegrated more rapidly than plasma-sprayed implants. We hypothesized that the accelerated osseointegration observed with the porous-surfaced design was the result of this design providing a local mechanical environment that was more favourable for bone formation. In the present study, we tested this hypothesis using finite element analysis and homogenization methods to predict the local strains in the pre-mineralized tissues formed around porous-surfaced and plasma-sprayed implants. We found that, for loading perpendicular to the implant interface, the porous surface structure provided a large region that experienced low distortional and volumetric strains, whereas the plasma-sprayed implant provided little local strain protection to the healing tissue. The strain protected region, which was within the pores of the sintered porous surface layer. corresponded to the region where the difference in the amount of mineralization between the two implant designs was the greatest. Low distortional and volumetric strains are believed to favour osteogenesis, and therefore the model results provide initial support for the hypothesis that the porous-surfaced geometry provides a local mechanical environment that favours more rapid bone formation in certain situations.

The effect of proximally and fully porous-coated canine hip stem design on bone modeling.

Porous coated canine femoral hip replacement implants were evaluated for biological fixation by bone ingrowth and the effect of the extent of porous coating on bone modeling. The Co-Cr alloy implants were either fully porous coated or coated only on the proximal 40% of the stem. Two implants of each type were studied 9, 16, and 36 months after surgery. Implant fixation and bone modeling were assessed radiographically throughout the implant periods and histologically after the test animals were killed. All 12 implants appeared stably fixed within the femur and were bone-ingrown in the porous region. Radiographic features such as proximal medial and anterior cortical thinning, proximal cancellous bone hypertrophy, and new endosteal bone formation near the stem tip were noted within the first postoperative year, with no appreciable change thereafter. The extent of proximal cortical thinning varied from virtually none to as much as 40%, being more prominent with the proximally coated implants at 16 months and with the fully coated implants at 36 months. Of consistent note was cancellous hypertrophy at the junction of porous and smooth implant surfaces with proximally coated implants and new endosteal bone formation and ingrowth at the stem tip of fully coated implants. These results indicate that the proximally porous-coated implant design causes increased proximal stress transfer, but this does not necessarily preclude proximal cortical resorption.

Characterization of nucleus pulposus-like tissue formed in vitro.

In order to be able to study the metabolism of nucleus pulposus (NP) tissue, we developed a cell culture system that resulted in the formation of NP-like tissue in vitro. NP cells were isolated from sheep lumbar spines and grown on filter inserts (Millicell CM). Histological examination showed that the cells accumulated extracellular matrix and formed a continuous layer of NP-like tissue. The accumulation of sulfated proteoglycans in the NP-like tissue continued up to 10 weeks and this was paralleled by an increase in tissue thickness and dry weight. DNA content remained stable during the first 4 weeks but then decreased over time. The amount of DNA, glycosaminoglycan (GAG) and collagen per mg dry weight of the tissue generated after 10 weeks in culture were 1.25+/-0.02, 301.6+/-27.7 and 411+/-65 microg, respectively, compared with 1.04+/-0.08, 320.6+/-21.2 and 399+/-4.4 microg (mean +/- SEM) for the in vivo tissue. There was no significant difference between in vitro and in vivo tissue. The cells in culture synthesized large proteoglycans (kav = 0.26+/-0.03, mean +/- S.D.) which were similar in size to those synthesized by cells in NP tissue in ex vivo culture (kav = 0.22+/-0.02, mean +/- S.D.) as determined by Sepharose CL-2B column chromatography. The in vitro generated tissue contained type II collagen as demonstrated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and silver staining as well as Western blot analysis. NP cells grown on filters generate tissue similar in composition to the in vivo tissue, for the characteristics examined to date, and should be a suitable model to use to study NP metabolism and extracellular matrix turnover.

Effects of dentin surface treatments on the fracture toughness and tensile bond strength of a dentin-composite adhesive interface.

It has been proposed that the fracture toughness test provides an appropriate method for assessing the fracture resistance of the dentin-composite interface. The plane-strain fracture toughness test was therefore applied to a dentin-composite interface, with use of a specific dentinal adhesive, so that the effects of various dentin surface treatments on dentin-bond integrity could be studied. Interfacial fracture toughness (KIC) values were determined following 24h and 180 days of specimen aging in distilled water at 37 degrees C. Tensile bond strength (TBS) results following 24-hour aging were also obtained for comparison with the 24-hour KIC results. In general, the fracture resistance of the dentin-composite interface was highest when the dentin surface was conditioned with acid but not air-dried, intermediate when the dentin surface was conditioned with acid and subsequently air-dried, and lowest when the dentin was not conditioned with acid. The tensile bond strength results differed from the fracture toughness results in indicating differences in surface preparation effects and the type of interfacial failure observed.

Fracture surface characterization of dentin-bonded interfacial fracture toughness specimens.

Although the current trend in dentin bonding favors the development of a hybrid layer interdiffusion zone for micromechanical bonding, the exact nature of the dentin-composite bond is still unclear. The objective of this study was to characterize the fracture surfaces of specimens used to measure interfacial fracture toughness. Morphological (SEM) and chemical (EDS and XPS) surface analyses were used for characterization. Fracture toughness specimens generally failed along the dentin-bonded interface in agreement with observed clinical failure modes. Four sites of bond failure were identified within the dentin-composite interfaces when All-Bond 2, Scotchbond Multi-Purpose, and Scotchbond 2 were used as the dentinal adhesives. These were located within (1) the smear layer, (2) a resin-modified layer between the interdiffusion zone and the adhesive resin, (3) a well-infiltrated hybrid interdiffusion zone, and (4) a non-infiltrated unsupported collagen layer. The interfacial region had a complex architecture which varied with the nature of the dentin, the dentin surface treatment, and the dentin bonding system. The sites of bond failure appeared to correlate with the interfacial fracture toughness and the extent to which polymerized resin infiltrated and acted to support the organic dentinal structures.

Fracture toughness of dentin/resin-composite adhesive interfaces.

The reliability and validity of tensile and shear bond strength determinations of dentin-bonded interfaces have been questioned. The fracture toughness value (KIC) reflects the ability of a material to resist crack initiation and unstable propagation. When applied to an adhesive interface, it should account for both interfacial bond strength and inherent defects at or near the interface, and should therefore be more appropriate for characterization of interface fracture resistance. This study introduced a fracture toughness test for the assessment of dentin/resin-composite bonded interfaces. The miniature short-rod specimen geometry was used for fracture toughness testing. Each specimen contained a tooth slice, sectioned from a bovine incisor, to form the bonded interface. The fracture toughness of an enamel-bonded interface was assessed in addition to the dentin-bonded interfaces. Tensile bond strength specimens were also prepared from the dentin surfaces of the cut bovine incisors. A minimum of ten specimens was fabricated for each group of materials tested. After the specimens were aged for 24 h in distilled water at 37 degrees C, the specimens were loaded to failure in an Instron universal testing machine. There were significant differences (p < 0.05) between the dental adhesives tested. Generally, both the fracture toughness and tensile bond strength measurements were highest for AllBond 2, intermediate for 3M MultiPurpose, and lowest for Scotchbond 2. Scanning electron microscopy of the fractured specimen halves confirmed that crack propagation occurred along the bond interface during the fracture toughness test. It was therefore concluded that the mini-short-rod fracture toughness test provided a valid method for characterization of the fracture resistance of the dentin-resin composite interface.

Fracture toughness of dental composites determined using the short-rod fracture toughness test.

Plane strain fracture toughness (KIC) has been evaluated for a number of commercially-available dental composites. A modified Short-rod Fracture Toughness (SRFT) specimen design has been used, enabling small specimens to be tested conveniently. The effect on KIC of aging in water at 37 degrees C for seven days, one month, and six months has been determined for conventional, microfilled, and hybrid (coarse and fine filler particle-containing) composites. Our results suggest that aging for one month or more caused a reduction of KIC for the composites so aged. Comparison of the KIC values determined using the modified SRFT specimen with values obtained using more conventional specimen geometries gave good agreement, thereby suggesting the suitability of the small SRFT specimens for valid KIC determinations.