A rapid seeding technique for the assembly of large cell/scaffold composite constructs.

These studies address critical technical issues involved in creating human mesenchymal stem cell (hMSC)/ scaffold implants for cartilage repair. These issues include obtaining a high cell density and uniform spatial cell distribution within the scaffold, factors that are critical in the initiation and homogeneity of chondrogenic differentiation. For any given scaffold, the initial seeding influences cell density, retention, and spatial distribution within the scaffold, which eventually will affect the function of the construct. Here, we discuss the development of a vacuum-aided seeding technique for HYAFF -11 sponges which we compared to passive infiltration. Our results show that, under the conditions tested, hMSCs were quantitatively and homogeneously loaded into the scaffolds with 90+% retention rates after 24 h in perfusion culture with no negative effect on cell viability or chondrogenic potential. The retention rates of the vacuum-seeded constructs were at least 2 times greater than those of passively seeded constructs at 72 h. Histomorphometric analysis revealed that the core of the vacuum-seeded constructs contained 240% more cells than the core of passively infiltrated scaffolds. The vacuum seeding technique is safe, rapid, reproducible, and results in controlled quantitative cell loading, high retention, and uniform distribution.

High-throughput aggregate culture system to assess the chondrogenic potential of mesenchymal stem cells.

We have developed an improved method for preparing cell aggregates for in vitro chondrogenesis studies. This method is a modification of a previously developed conical tube-based culture system that replaces the original 15-mL polypropylene tubes with 96-well plates. These modifications allow a high-throughput approach to chondrogenic cultures, which reduces both the cost and time to produce chondrogenic aggregates, with no detrimental effects on the histological and histochemical qualities of the aggregates. We prepared aggregates in both systems with human bone marrow-derived mesenchymal stem cells (hMSC). The aggregates were harvested after 2 and 3 weeks in chondrogenic culture and analyzed for their ability to differentiate along the chondrogenic pathway in a defined in vitro environment. Chondrogenic differentiation was assessed biochemically by DNA and glycosaminoglycan (GAG) quantification assays and by histological and immunohistologic assessment. The chondrogenic cultures produced in the 96-well plates appear to be slightly larger in size and contain more DNA and GAG than the aggregates made in tubes. When analyzed histologically, both systems demonstrate morphological characteristics that are consistent with chondrogenic differentiation and cartilaginous extracellular matrix production.

Bioreactors mediate the effectiveness of tissue engineering scaffolds.

We hypothesized that the mechanically active environment present in rotating bioreactors mediates the effectiveness of three-dimensional (3D) scaffolds for cartilage tissue engineering. Cartilaginous constructs were engineered by using bovine calf chondrocytes in conjunction with two scaffold materials (SM) (benzylated hyaluronan and polyglycolic acid); three scaffold structures (SS) (sponge, non-woven mesh, and composite woven/non-woven mesh); and two culture systems (CS) (a bioreactor system and petri dishes). Construct size, composition [cells, glycosaminoglycans (GAG), total collagen, and type-specific collagen mRNA expression and protein levels], and mechanical function (compressive modulus) were assessed, and individual and interactive effects of model system parameters (SM, SS, CS, SM*CS and SS*CS) were demonstrated. The CS affected cell seeding (higher yields of more spatially uniform cells were obtained in bioreactor-grown than dish-grown 3-day constructs) and subsequently affected chondrogenesis (higher cell numbers, wet weights, wet weight GAG fractions, and collagen type II levels were obtained in bioreactor-grown than dish-grown 1-month constructs). In bioreactors, mesh-based scaffolds yielded 1-month constructs with lower type I collagen levels and four-fold higher compressive moduli than corresponding sponge-based scaffolds. The data imply that interactions between bioreactors and 3D tissue engineering scaffolds can be utilized to improve the structure, function, and molecular properties of in vitro-generated cartilage.

Performance of polyoxymethylene plastic (POM) as a component of a tissue engineering bioreactor.

Polyoxymethylene (POM, acetal homopolymer, polyacetal), commercialized as Delrin by DuPont, is an engineering resin with mechanical properties that make it useful for the prototyping and manufacture of laboratory apparatus. These properties include excellent, "metal-like," machining characteristics and dimensional stability, as well as thermal stability, which allows steam sterilization. Historically, POM has been used widely, including as a surgical implant material. For these reasons, we have used this plastic as a media-wetted component in a tissue-engineering bioreactor, with good results. However, a study by LaIuppa et al.5 suggested that POM is unsuitable for use in a cell culture environment (LaIuppa et al. J Biomed Mater Res 1997;36:347-359). POM is based on the polymerization of formaldehyde, and, in addition, contains stabilizers and/or fillers. All of these could potentially be released into the medium, e.g., as formaldehyde or other thermal breakdown products, especially upon repeated autoclaving. The cited report thus appeared plausible, although contrary to our observations. In this study, we specifically assessed whether media conditioned by long-term exposure to machined white POM had a negative effect on the proliferation and chondrogenic differentiation of human mesenchymal stem cells (MSCs). We selected this cell system, as cartilage tissue engineering is the primary application of our bioreactor system. The POM samples were steam-autoclaved 1 to 20 times, to assess the possibility of any toxic thermal breakdown product release into the media. We found that MSCs did not attach directly to machined POM. Because cells that escape from the tissue construct cannot colonize the reactor and compete for nutrients, this is a desirable characteristic of a material used in a tissue-engineering bioreactor. Furthermore, the use of POM-conditioned media had no detectable impact on the proliferation rate of MSCs measured over a one-week period; nor was any effect on chondrogenic differentiation observed at up to 3 weeks in culture. In summary, the use of POM as a culture medium-wetted component appears to be innocuous, at least for human MSCs. The contrast of these findings to those of LaIuppa et al.5 may reflect a cell-type specific sensitivity, or may be due to different handling of the material.

Repair of osteochondral defect with tissue-engineered two-phase composite material of injectable calcium phosphate and hyaluronan sponge.

Articular cartilage has limited capacity for repair. In the present study, tissue-engineered two-phase composite material was used for the repair of osteochondral defects in young adult rabbit knee. This composite material is composed of an injectable calcium phosphate (ICP) and a hyaluronan (HA) derivate of either ACP or HYAFF 11 sponge. The osteochondral defect, 3 mm in diameter and 3 mm deep, was created in the weight-bearing region of the medial femoral condyle. The bone portion of the defect was first filled with ICP to a level approximately 1 mm below the articular surface. HA sponge (3 mm in diameter and 1-1.2 mm thick), with or without loading of autologous bone marrow-derived progenitor cells (MPCs), was then inserted into the defect on top of the ICP as it hardened. Animals were allowed free cage activity postoperatively, and killed 4 or 12 weeks (for the HYAFF 11 sponge group) after the surgery. At 4 weeks, histological examination showed that the defect was filled up to 90-100% of its depth. Whitish repair tissue on the top appeared to be integrated with the surrounding articular cartilage. Four distinct zones of repair tissue were identified: a superficial layer, a chondroid tissue layer, an interface between HA sponge and ICP, and the ICP material. Evidence of extensive osteoclastic and osteoblastic activities was observed in the bone tissue surrounding the defect edge and in ICP material. By 12 weeks, the zonal features of the repair tissue became more distinct; chondrocytes were arranged in a columnar array, and a calcified layer of cartilage was formed beneath the chondroid tissue in some specimens. The healing tissue of the HA sponge material loaded with MPCs had higher cellular density and better integration with the surrounding cartilage than HA sponge material not loaded with MPCs. This study suggests that using a two-phase composite graft may hold potential for the repair of osteochondral defects by providing mechanical support that mimicks subchondral bone, while also providing a chondrogenic scaffold for the top cartilage repair.

Treatment of osteochondral defects with autologous bone marrow in a hyaluronan-based delivery vehicle.

The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable and bioactive materials that provide structural support and molecular cuing to stimulate repair. Since bone marrow contains osteochondral progenitor cells and bioactive agents, it is hypothesized that the combination of scaffold and bone marrow would be a superior composite material for osteochondral repair. This hypothesis will be tested by comparing the outcome of osteochondral defects filled with a fibronectin-coated hyaluronan-based sponge (ACP) with or without autologous bone marrow. Thirty-three 4-month-old rabbits received 3-mm diameter osteochondral defects that were then filled with ACP loaded or not with autologous bone marrow. Rabbits were sacrificed at 2, 3, 4, 12, and 24 weeks after surgery and the condyles processed for histologic and immunohistochemical evaluation. The defects were graded with a histologic scoring scale. Except for the 3-week specimens, the histologic appearance of the defects was similar in both groups. Four weeks after surgery, the defects were filled with bone with a top layer of cartilage well integrated with the adjacent cartilage. Twelve and 24 weeks after surgery, the defects again showed bone filling. The primary difference between the 4-week samples and the 12- and 24-week samples was that the layer of cartilage that appeared to be thinner than the adjacent cartilage. At each harvest time, the overall histologic scores of the specimens did not reveal statistical differences between the treatment groups. However, as revealed by the results of the 3-week sacrifices, bone marrow loading appeared to accelerate the first stages of the repair process. The fibronectin-coated hyaluronan-based scaffold appears to organize the natural response and facilitate the integration of the neo-cartilage with the adjacent tissue. The fundamental tissue engineering principles derived from this study should provide guidelines for the development of comparable clinical reconstructive therapies.

Controlled release of hyaluronan oligomers from biodegradable polymeric microparticle carriers.

In the present study, biodegradable microparticles of blends of poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) were explored as a potential carrier for the controlled release of polysaccharide oligomers. To this end, hyaluronan (HY) oligomers of varying molecular weights were incorporated into PLGA/PEG microparticles. Using a two-level fractional factorial experimental design, four microparticle formulation parameters, the amount of PEG included in the microparticles, the initial HY loading of the microparticles, the molecular weight of HY, and the molecular weight of PLGA, were studied for their influence on the incorporation and in vitro release of HY over the period of 28 days. The entrapment efficiencies were found to range between 10+/-1% and 24+/-2% depending on the initial loading and the molecular weight of the HY oligomer used in the fabrication of the microparticles. The HY was released in a multiphasic fashion including an initial burst release, followed by two separate periods of linear release. The normalized cumulative mass released during the burst release ranged from 25.1+/-9.2% to 93.0+/-0.7% and was found to be significantly influenced by the initial HY loading, the HY molecular weight, and the PLGA molecular weight. The initial period of linear release lasted from day 1 to day 14 and displayed normalized cumulative rates of release from 0.1+/-0.0%/day to 1.4+/-0.2%/day. During this period, PEG content of the microparticles and HY molecular weight exerted the greatest influence on the rate of release. Finally, the second period of linear release lasted through the final time-point at day 28. Here, the normalized cumulative rate of release values ranged from 0.2+/-0.1%/day to 3.6+/-0.7%/day and were dependent on all formulation parameters studied. These results demonstrate the potential of PLGA/PEG blend microparticles for the controlled release of HY oligomers.

Effect of implantation of Augment(®) Bone Graft on serum concentrations of platelet-derived growth factors: a pharmacokinetic study.

BACKGROUND: Augment(®) Bone Graft is a bone graft substitute intended to be used as an alternative to autologous bone graft in the fusion of hindfoot and ankle joints. Augment(®) Bone Graft is a combination device comprised of beta-tricalcium phosphate (ß-TCP) and recombinant human platelet-derived growth factor BB homodimer (rhPDGF-BB). OBJECTIVE: This human pharmacokinetic study was undertaken to assess the effect of Augment(®) Bone Graft implantation on the serum concentration of platelet-derived growth factors (PDGFs). METHODS: Under the terms of a Research Ethics Board-approved protocol, Augment(®) Bone Graft was implanted in patients (n = 7) undergoing hindfoot and ankle arthrodesis procedures requiring graft material. The control cohort of the study (n = 4) received autologous bone graft. The serum concentrations of PDGF isoforms AA, AB and BB in blood samples, obtained prior to and at ten time points (up to 7 days) after surgery, were measured using enzyme-linked immunosorbent assays (ELISA). RESULTS: The serum concentration of PDGF-BB did not vary significantly from baseline (median of the combined cohorts 3.89 ng/mL) throughout the course of the study. The serum concentrations of PDGF-AA, PDGF-AB and total PDGF did not deviate from their baseline values (medians of the combined cohorts were 2.87, 14.95 and 20.19 ng/mL for PDGF-AA, PDGF-AB and total PDGF, respectively) except for the last time point in which they were increased (medians for the combined cohorts were 4.71, 20.42 and 30.29 ng/mL for PDGF-AA, PDGF-AB and total PDGF, respectively). There were no differences between the two treatment groups with regard to changes in the serum concentrations of PDGF. None of the samples tested contained anti-PDGF-BB antibodies. CONCLUSION: Analysis of the data demonstrated that the serum concentrations of all three PDGF isoforms analysed were unaffected by implantation of Augment(®) Bone Graft.

The role of recombinant human platelet-derived growth factor-BB (rhPDGF-BB) in orthopaedic bone repair and regeneration.

Recombinant human PDGF BB homodimer (rhPDGF-BB) is a potent recruiter of, and strong mitogenic factor for, cells crucial to musculoskeletal tissue repair, including mesenchymal stem cells (MSCs), osteogenic cells and tenocytes. rhPDGF-BB also upregulates angiogenesis. These properties allow rhPDGF-BB to trigger the cascade of bone and adjoining soft tissue repair and regeneration. This mechanism of action has been established in numerous preclinical and clinical studies. Demonstration of the safety and efficacy of rhPDGF-BB in the healing of chronic foot ulcers in diabetic patients and regeneration of alveolar (jaw) bone lost due to chronic infection from periodontal disease has resulted in two FDA-approved products based on this molecule. A third product is in late stages of clinical development, with pilot and pivotal clinical studies of rhPDGF-BB mixed with an osteoconductive bone matrix (Augment(®) Bone Graft) in foot and ankle fusions demonstrating that this product is at least as effective as bone autograft, and has an improved safety profile. Additional combinations of rhPDGF-BB with tissue-specific matrices are also being studied clinically in additional musculoskeletal indications.

Repair of osteochondral defects with hyaluronan- and polyester-based scaffolds.

OBJECTIVE: The natural repair of osteochondral defects can be enhanced with biocompatible, biodegradable materials that support the repair process. It is our hypothesis that hyaluronan-based scaffolds are superior to synthetic scaffolds because they provide biological cues. We tested this thesis by comparing two hyaluronan-based scaffolds [auto cross-linked polysaccharide polymer (ACP) and HYAFF-11] to polyester-based scaffolds [poly(DL-lactic-co-glycolic acid) (PLGA) and poly(L-lactic acid) (PLLA)] with similar pore size, porosity and degradation times. DESIGN: Fifty-four rabbits received bilateral osteochondral defects. One defect received a hyaluronan-based scaffold and the contralateral defect received the corresponding polyester-based scaffold. Rabbits were euthanized 4, 12 and 20 weeks after surgery and the condyles dissected and processed for histology. RESULTS: Only ACP-treated defects presented bone at the base of the defect at 4 weeks. At 12 weeks, only defects treated with rapidly dissolving implants (ACP and PLGA) presented bone reconstitution consistently, while bone was present in only one third of those treated with slowly dissolving scaffolds (HYAFF-11 and PLLA). After 20 weeks, the articular surface of PLGA-treated defects presented fibrillation more frequently than in ACP-treated defects. The surface of defects treated with slowly dissolving scaffolds presented more cracks and fissures. CONCLUSIONS: The degradation rate of the scaffolds is critical for the repair process. Slowly dissolving scaffolds sustain thicker cartilage at the surface but, it frequently presents cracks and discontinuities. These scaffolds also delay bone formation at the base of the defects. Hyaluronan-based scaffolds appear to allow faster cell infiltration leading to faster tissue formation. The degradation of ACP leads to rapid bone formation while the slow degradation of HYAFF-11 prolongs the presence of cartilage and delays endochondral bone formation.