Marsha S. Reuther

In vivo implantation of tissue-engineered human nasal septal neocartilage constructs: a pilot study.

Objective. To determine the in vivo biocompatibility of septal neocartilage constructs developed in vitro by an alginate intermediate step. Study Design. Prospective, animal model. Setting. Research laboratory. Subjects and Methods. A murine model was used to examine the maturation of neocartilage constructs in vivo. Chondrocytes collected from patients undergoing septoplasty were expanded in monolayer and suspended in alginate beads for 3-dimensional culture in media containing human serum and growth factors. After in vitro incubation for 5 weeks, the constructs were implanted in the dorsum of athymic mice for 30 and 60 days (n = 9). After the mice were sacrificed, the constructs were recovered for assessment of their morphological, histochemical, biochemical, and biomechanical properties. Results. The mice survived and tolerated the implants well. Infection and extrusion were not observed. Neocartilage constructs maintained their general shape and size and demonstrated cell viability after implantation. The implanted constructs were firm and opaque, sharing closer semblance to native septal tissue relative to the gelatinous, translucent preimplant constructs. Histochemical staining with hematoxylin and eosin (H&E) revealed that the constructs exhibited distinct morphologies characteristic of native tissue, which were not observed in preimplant constructs. DNA and type II collagen increased with duration of implantation, whereas type I collagen and glycoaminoglycans (GAG) decreased. Mechanical testing of a 60-day implanted construct demonstrated characteristics similar to native human septal cartilage. Conclusions. Neocartilage constructs are viable in an in vivo murine model. The histologic, biochemical, and biomechanical features of implanted constructs closely resemble native septal tissue when compared with preimplant constructs.

Scar revision techniques-pearls and pitfalls.

Although scars are a normal part of the healing process, facial scars have significant implications on a patients well-being, both physically and psychologically. Facial scars are one of the most common reasons a patient presents to the facial plastic and reconstructive surgeon. The ability to evaluate facial scars and choose the most appropriate technique for revision is of paramount importance to obtain the best result. A thorough understanding of superficial facial anatomy and principles of wound healing is equally as important as meticulous technical execution. Above all, the expectations of the patient must be assessed and considered when formulating a surgical plan.

Tissue-engineered cartilage for facial plastic surgery.

Purpose of reviewThe reconstruction of cartilaginous craniofacial defects is ideally performed with analogous grafting material, such as autologous tissue. However, the use of autologous cartilage is limited by its finite availability and potentially suboptimal geometry to repair specific defects. Tissue engineering of human cartilage may provide the adequate supply of grafting and implant material for the reconstruction of cartilaginous facial defects. An update of the various cartilage tissue engineering methodologies is provided in this review. Recent findingsThe cartilage tissue engineering paradigm begins with the harvest of a small septal cartilage donor specimen. This is followed by the isolation and subsequent proliferation of chondrocytes and the seeding of these cells onto three-dimensional scaffolds. Neocartilage is created as pericellular substrate, is produced by the cells and deposited throughout the scaffold. Theoretically, the mature cartilage construct can be introduced back into the same patient for reconstruction of craniofacial defects. Initial steps of the cartilage tissue engineering protocol have been standardized; however, modifications of subsequent steps have shown the potential to profoundly impact tissue composition and strength, bringing the properties of cartilage constructs closer to those of native human septum. SummaryThe ability to engineer virtually limitless quantities of autologous cartilage could have a profound impact on facial plastic and reconstructive surgery. The strategies used to refine human cartilage culture techniques have successfully produced neocartilage constructs with biochemical and biomechanical properties approaching those of native septal tissue. With the steady progress achieved in recent years, there is great capacity for the proximate realization of surgically implantable tissue-engineered cartilage constructs.

In vivo oxygen tension in human septal cartilage increases with age.

Tissue‐engineered septal cartilage may provide a source of autologous cartilage for repair of nasal defects. Production of clinically useful neocartilage involves multiple steps that include manipulating the culture environment. Partial pressure of oxygen (ppO2) is a property that has been shown to influence cartilage development. Specifically, studies suggest low ppO2 augments in vitro growth of articular cartilage. Although in vivo measurements of articular cartilage ppO2 have demonstrated hypoxic conditions, measurements have not been performed in septal cartilage. The objective of this study was to determine the ppO2 of septal cartilage in vivo.

Culture of Human Septal Chondrocytes in a Rotary Bioreactor

Objectives (1) To show that extracellular matrix deposition in 3-dimensional culture of human septal chondrocytes cultured in a rotary bioreactor is comparable to the deposition achieved under static culture conditions. (2) To demonstrate that the biomechanical properties of human septal chondrocytes cultured in a bioreactor are enhanced with time and are analogous to beads cultured under static culture. Study Design Prospective, basic science. Setting Research laboratory. Methods Human septal chondrocytes from 9 donors were expanded in monolayer and seeded in alginate beads. The beads were cultured in a rotary bioreactor for 21 days in media supplemented with growth factors and human serum, using static culture as the control. Biochemical and biomechanical properties of the beads were measured. Results Glycosaminoglycan (GAG) accumulation significantly increased during 2 measured time intervals, 0 to 21 days and 10 to 21 days (P < .01). No significant difference was seen between the static and bioreactor conditions. Substantial type II collagen production was demonstrated in the beads terminated at day 21 of culture in both conditions. In addition, the biomechanical properties of the beads were significantly improved at 21 days in comparison to beads from day 0. Conclusion Human septal chondrocytes cultured in alginate beads exhibit significant matrix deposition and improved biomechanical properties after 21 days. Alginate bead diameter and stiffness positively correlated with GAG and type II collagen accretion. Matrix production in beads is supported by the use of a rotary bioreactor.

Effect of oxygen tension on tissue-engineered human nasal septal chondrocytes.

Tissue-engineered nasal septal cartilage may provide a source of autologous tissue for repair of craniofacial defects. Although advances have been made in manipulating the chondrocyte culture environment for production of neocartilage, consensus on the best oxygen tension for in vitro growth of tissue-engineered cartilage has not been reached. The objective of this study was to determine whether in vitro oxygen tension influences chondrocyte expansion and redifferentiation. Proliferation of chondrocytes from 12 patients expanded in monolayer under hypoxic (5% or 10%) or normoxic (21%) oxygen tension was compared over 14 days of culture. The highest performing oxygen level was used for further expansion of the monolayer cultures. At confluency, chondrocytes were redifferentiated by encapsulation in alginate beads and cultured for 14 days under hypoxic (5 or 10%) or normoxic (21%) oxygen tension. Biochemical and histological properties were evaluated. Chondrocyte proliferation in monolayer and redifferentiation in alginate beads were supported by all oxygen tensions tested. Chondrocytes in monolayer culture had increased proliferation at normoxic oxygen tension (p = 0.06), as well as greater accumulation of glycosaminoglycan (GAG) during chondrocyte redifferentiation (p < 0.05). Chondrocytes released from beads cultured under all three oxygen levels showed robust accumulation of GAG and type II collagen with a lower degree of type I collagen immunoreactivity. Finally, formation of chondrocyte clusters was associated with decreasing oxygen tension (p < 0.05). Expansion of human septal chondrocytes in monolayer culture was greatest at normoxic oxygen tension. Both normoxic and hypoxic culture of human septal chondrocytes embedded in alginate beads supported robust extracellular matrix deposition. However, GAG accumulation was significantly enhanced under normoxic culture conditions. Chondrocyte cluster formation was associated with hypoxic oxygen tension.

Shape Fidelity of Native and Engineered Human Nasal Septal Cartilage

Objective To test engineered and native septal cartilage for resistance to deformation and remodeling under sustained bending loads and to determine the effect of bending loads on the biochemical properties of constructs. Study Design Prospective, basic science. Setting Laboratory. Subjects and Methods Human septal chondrocytes from 6 donors were used to create 12-mm constructs. These were cultured for 10 weeks and subjected to bending for 6 days. Free-swelling controls and native tissue from 6 donors were used for comparison. Shape retention, photo documentation, live-dead staining, and biochemical properties were measured. Results Live-dead staining showed no difference in cell survival between loaded constructs and free-swelling controls. The immediate shape retention of the constructs was 39.0% versus 24.4% for native tissue (P = .13). After 2 and 24 hours of relaxation, the constructs possessed similar shape retention to native tissue (26.9% and 16.4%; P = .126; 21.7% and 14.4%; P = .153). There was no significant change in construct shape retention from immediately after release to 2 hours of relaxation (39.0% and 26.9%, respectively; P = .238). In addition, the retention did not change significantly between 2 and 24 hours of relaxation (26.9% and 21.7%; P = .48). There was no significant difference in biochemical properties between loaded constructs and controls. Conclusion The shape retention properties of human septal neocartilage constructs are comparable to human native septal cartilage. In addition, mechanical loading of neocartilage constructs does not adversely affect cell viability or biochemical properties. This study demonstrates that neocartilage constructs possess adequate shape fidelity for use as septal cartilage graft material.

Tissue Engineering and the Future of Facial Volumization

Volume loss due to facial aging can be restored by facial volumization using a variety of materials. Volumization can be performed in isolation or concurrent with other facial rejuvenation procedures to obtain an optimal aesthetic result. There is a myriad of manufactured products available for volumization. The use of autologous fat as facial filler has been adopted more recently and possesses certain advantages; however, the ideal filler is still lacking. Tissue engineering may offer a solution. This technology would provide autologous soft-tissue components for use in facial volumization. The use of stem cells may enable customization of the engineered product for the specific needs of each patient.

Volume Expansion of Tissue Engineered Human Nasal Septal Cartilage.

IMPORTANCE Cartilaginous craniofacial defects range in size and autologous cartilaginous tissue is preferred for repair of these defects. Therefore, it is important to have the ability to produce large size cartilaginous constructs for repair of cartilaginous abnormalities. OBJECTIVES To produce autologous human septal neocartilage constructs substantially larger in size than previously produced constructsTo demonstrate that volume expanded neocartilage constructs possess comparable histological and biochemical properties to standard size constructsTo show that volume expanded neocartilage constructs retain similar biomechanical properties to standard size constructs. DESIGN Prospective, basic science. SETTING Laboratory. PARTICIPANTS The study used remnant human septal specimens removed during routine surgery at the University of California, San Diego Medical Center or San Diego Veterans Affairs Medical Center. Cartilage from a total of 8 donors was collected. MAIN OUTCOMES MEASURED Human septal chondrocytes from 8 donors were used to create 12mm and 24mm neocartilage constructs. These were cultured for a total of 10 weeks. Photo documentation, histological, biochemical, and biomechanical properties were measured and compared. RESULTS The 24mm diameter constructs were qualitatively similar to the 12mm constructs. They possessed adequate strength and durability to be manually manipulated. Histological analysis of the constructs demonstrated similar staining patterns in standard and volume expanded constructs. Proliferation, as measured by DNA content, was similar in 24mm and 12mm constructs. Additionally, glycosaminoglycan (GAG) and total collagen content did not significantly differ between the two construct sizes. Biomechanical analysis of the 24mm and 12mm constructs demonstrated comparable compressive and tensile properties. CONCLUSION AND RELEVANCE Volume expanded human septal neocartilage constructs are qualitatively and histologically similar to standard 12mm constructs. Biochemical and biomechanical analysis of the constructs demonstrated equivalent properties. This study shows that modification of existing protocols is not required to successfully produce neocartilage constructs in larger sizes for reconstruction of more substantial craniofacial defects. LEVEL OF EVIDENCE NA.

Advancing the Art of Rhinoplasty with Tissue Engineering

Tissue engineering of human septal cartilage could profoundly affect our surgical decisions with primary and revision rhinoplasties. This technology would provide adequate quantity of engineered tissue for cartilage grafting that is frequently used in nasal reconstruction. In theory, the process would begin after a small septal cartilage donor specimen is obtained from the patient. Many of the initial steps in preparing the tissue involve standardized tissue culture techniques. However, modifications to the subsequent tissue growth process tend to affect the success for correct tissue development and maturation. These modifications have a significant impact on the tissue composition and its mechanical strength, which is critical in engineering a cartilage graft suitable for implantation into the nasal framework. An update of these various tissue growth methodologies is provided in this chapter.