WO2016065254A1 - Compositions and methods for cartilage replacement - Google Patents

Abstract

Compositions and methods for the synthesis of cartilage are disclosed.

Description

COMPOSITIONS AND METHODS FOR CARTILAGE REPLACEMENT

This application claims priority under 35 U.S.C. § 1 19(e) to U.S. Provisional Patent Application No. 62/067,681, filed October 23, 2014. The foregoing application is incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to the field of cartilage replacement therapy. More specifically, the invention provides compositions and methods for the synthesis of improved cartilage.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout the specification in order to describe the state of the art to which this invention pertains. Each of these citations is incorporated herein by reference as though set forth in full.

The mainstay for the repair of severe subglottic stenosis is laryngotracheal reconstruction (LTR) with autologous cartilage grafts harvested from the rib, larynx or ear. LTR expands the laryngotracheal airway with elliptically shaped free cartilage grafts placed in the anterior position, posterior position or both (Cotton, R.T. (1991)

Laryngoscope 101 (Suppl 56): 1-34). These techniques carry a high success rate, but a significant percentage fail first time reconstructive surgery and require additional approaches (Hartnick et al. (2001) Ann. Otol. Rhinol. LaryngoL, 1 10: 1109-11 13; Rizzi et al. (2009) Otolaryngol. Head Neck Surg., 140:348-53). Shortcomings of the standard approaches include the potential for restenosis, limitations in the available size, and shape of available cartilage, and resorption of free cartilage grafts. In fact, it has been established that 39% of autologous cartilage resorbs at 10 weeks after LTR in a rabbit model (Jacobs et al. (1999) Ann. Otol. LaryngoL Rhinol., 108:599-605). This shrinkage may result in restenosis of the airway.

It would be ideal to provide a customized graft with desired shape, size, and rigidity that would match laryngeal cartilage and allow for more effective reconstructive techniques. Tissue-engineering techniques have the potential to resolve these issues and handle more challenging problems such as total tracheal stenosis or agenesis. The greatest challenge is to create constructs that exhibit the proper strength, flexibility, as well as cellular and biochemical composition of hyaline cartilage which is composed of chondrocytes, water, collagen and glycosaminoglycans (GAG).

A great deal of work has been done during the last decade in regenerative medicine. Tissue-engineered constructs have been used to replace a variety of native tissue and organs including skin, blood vessels, heart and liver (Langer et al. (1993) Science 260:920-6). Cartilage tissue-engineering techniques have also evolved dramatically in the last 10-15 years based upon the use of synthetic scaffolds, load- bearing devices and tissue mimetic bioreactors. Chondrogenesis has been shown to be enhanced using both soluble growth factors such as TGF- 3 (Mauck et al. (2000) J. Biomech. Eng., 122:252-60; Bian et al. (2011) Biomaterials 32:6425-34) and mechanical stimulation such as dynamic compression (Kim et al. (2012) J. Mech. Behav. Biomed. Mater., 1 1 :92-101 ; Huang et al. (2010) Eur. Cell Mater., 19:72-85; Butler et al. (2000) J. Biomech. Eng., 122:570-5; Mauck et al. (2002) Ann. Biomed. Eng., 30: 1046-1056). However, even with this enhancement the tissue-engineered results only approximate native tissues with GAG content only approaching 40% and dynamic modulus of 20% (Butler et al. (2000) J. Biomech. Eng., 122:570-5; Mauck et al. (2002) Ann. Biomed. Eng., 30: 1046-1056; Anderson et al. (2004) Science 305: 1923-1924). Moreover, the laryngotracheal complex requires a distinct form of cartilage. First, LTR grafts require high mechanical strength and rigidity to resist internal forces leading to scar contracture in contrast to a compressible hyaline cartilage that is needed to bear weight such as in the knee joint. Soft materials would allow the airway to collapse. Novel synthetic scaffold materials should be designed with sufficient stiffness to resist deformational forces and remain in place, yet with enough porosity to allow the influx of cells, nutrients and oxygen (Huang et al. (2010) Eur. Cell Mater., 19:72-85; Butler et al. (2000) J. Biomech. Eng., 122:570-5). The second requirement for a cartilage graft in the laryngotracheal airway is long-term durability and survival. The tissue-engineered cartilage must be able to survive a lifetime in the airway without a major vascular supply, and must grow with the child. The third requirement is that the tissue-engineered cartilage should not elicit a significant inflammatory reaction. These features are essential for a biomaterial used to reconstruct the pediatric airway.

Preliminary work in cartilage tissue-engineering for the airway examined tissue- engineered constructs cultured in perfusion bioreactors with autologous chondrocytes from three sources: auricular, articular, and nasal. The auricular chondrocytes yielded the best results in terms of GAG content, type II collagen and mechanical strength with modulus of 0.21 MPa at 3 weeks diminishing to 0.100 MPa at 6 weeks (Henrickson et al. (2007) Tissue Eng., 13:843-53) while normal septal cartilage vertical compressive modulus is 0.7 MPa (Richmon et al. (2006) Am. J. Rhinol, 20:496-501). The modulus is an indicator of the tissue's response to force and offers insight into the stability, flexibility and ultimate feasibility of the tissue-engineered cartilage grafts. These results support the use of auricular chondrocytes, but the long-term sustainability of mechanical strength is essential for the pediatric airway.

While much work has been done in tracheal tissue-engineering, only a few studies have addressed tissue-engineered grafts for laryngotracheal reconstruction. Successful LTR in a piglet model was performed using human chondrocytes seeded in vivo for 8 weeks in P-27 polymeric scaffolds. This reconstruction was done with a normal piglet airway and material properties were not measured. Nevertheless, the grafts remained intact in the airway for 3 months (Kamil et al. (2004) Arch. Otolaryngol. Head Neck Surg., 130: 1048-1051). Hyalograft C has been used as a scaffold for LTR in a rabbit model and a severe inflammatory reaction to the scaffold was found with complete degradation (Weidenbecher et al. (2007) Laryngoscope 117: 1745-9). Rabbit LTR has also been performed using scaffold-free tissue engineered grafts. While none of the seven rabbits developed respiratory distress or inflammatory reactions, the tissue-engineered cartilage resulted in mechanical failure of the graft (Gilpin et al. (2010) Laryngoscope 120:612-617). In view of the going, improved compositions and methods for the synthesis of cartilage are needed. SUMMARY OF THE INVENTION

In accordance with the instant invention, methods of synthesizing cartilage are provided. The method comprises culturing chondrocytes in vitro in the presence of at least one chondrogenesis factor for sufficient time to synthesize a cartilage construct and then culturing the obtained cartilage construct in an animal host for maturation of the cartilage construct. The methods may further comprise isolating the cartilage construct from the animal host, particularly a human. In a particular embodiment, the chondrocytes are cultured in vitro with a scaffold, particularly one comprising hyaluronic acid. In a particular embodiment, the cartilage construct is cultured in an animal host by being inserted subcutaneously into the host. In a particular embodiment, the chondrocytes are cultured in vitro for at least about 12 weeks and maintained within the animal host for at least about 12 weeks. The instant invention also encompasses cartilage constructs obtained by the synthetic methods of the instant invention.

In accordance with another aspect of the instant invention, methods of replacing damaged or deficient cartilage in a subject are provided. The method comprises inserting a cartilage construct (e.g., graft) of the instant invention into the subject. In a particular embodiment, the chondrocytes used to synthesize the cartilage construct are autologous. The cartilage can be used to replace cartilage anywhere in the subject including the ear, nose, joints, knee, rib cage, larynx, trachea, and vertebrae.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A shows a 4x4 mm pilot construct undergoing compression testing. Figure IB shows an anterior-posterior compression test. Figure 1C shows a three-point bend test for flectoral modulus.

Figure 2A provides a lateral view of 1% hyaluronic acid (HA) construct after 12 weeks in vitro showing gross augmentation in volume due to production of extra-cellular matrix. The dimensions increase beyond 13 x 5 x 2.25 mm. Figure 2B shows four 1% HA after 12 weeks in vivo and 12 weeks in vitro. Figure 2C shows a 1% HA graft carved as a suitable LTR graft. Figure 2D shows the insertion of the graft.

Figures 3A, 3C, and 3E show tissue-engineered laryngotracheal reconstruction

(TE-LTR) using a 1% HA graft, using a 1% HA graft on a different rabbit, and using a control graft of thyroid alar cartilage, respectively. Figures 3B, 3D, and 3F show an endoscopic view 12 weeks after the laryngotracheal reconstruction of the rabbits in Figures 3A, 3C, and 3E, respectively.

Figure 4 provides a graph of the time course for compressive modulus of 4x4 mm discs using 1% HA, 2% HA and PGA-calcium alginate at 4, 8, and 12 week in vitro both in a rotary bioreactor (stripped columns) and static culture (solid columns).

Figure 5A shows a post-mortem examination of larynx of rabbit s/p PGA-calcium alginate cartilage graft that died of airway obstruction. Lumen reveals a large granuloma that obstructed the airway. Figure 5B provides a cross section of the airway stained with Safranin O revealing nearly complete obstructive granuloma (20x). Figure 5C shows a collagen II immune-stain of graft revealing PGA filaments (50x). Figure 5D shows a safranin O stain of graft revealing decrease in central cells and microfilaments (50x). Figure 5E shows a bony graft sutured into the airway. Figure 5F shows a gross post- mortem view revealing collapsed graft. Figure 5G shows a cross section of the airway stained with Safranin O revealing nearly complete obstructive collapsed graft (20x). Figure 5H shows a Safranin O stain of bony graft (50x).

Figures 6A-6D provide graphs of the flexural modulus of various constructs. Individual values are presented by groups. Bar represents mean with 95% confidence interval. Figure 6A shows moduli of HA vs. PGA constructs after 12 weeks in vitro. Figure 6B shows moduli of HA vs. PGA constructs after 12 weeks in vivo compared to control thyroid cartilage. Figure 6C shows moduli of HA constructs after 12 weeks in vitro vs. HA constructs after 12 weeks in vivo. Figure 6D shows moduli of PGA constructs after 12 weeks in vitro vs. 12 weeks in vivo.

Figures 7A and 7B show cricoid compressive moduli. The scatterplots show the range, mean (bar) and 95% confidence intervals. Figure 7A shows the anterior-posterior compressive moduli of TE-LTR vs. C-LTR (p = 0.11 1) and cricoid controls

(p = 0.004). Figure 7B shows the medial-lateral compressive moduli of TE-LTR vs. C- LTR (p = 0.999) and cricoid controls (p = 0.047).

Figures 8A-8C provide graphs of the glycosaminoglycans of constructs.

Individual values are presented by groups. Bar represents mean with 95% confidence interval. Figure 8A shows GAG of HA vs. PGA 12 in vitro and in vivo compared to control cartilage and negative control. Two high extreme control cartilage values are not shown as they are high on the Y-axis. Figure 8B shows GAG of HA after 12 weeks in vitro vs. after 12 weeks in vivo. Figure 8C shows GAG of PGA constructs after 12 weeks in vitro vs. after 12 weeks in vivo.

Figures 9A-9C provide graphs of GAG determination of the TE grafts vs.

controls. Figure 9A shows GAG content (% w/w) of constructs of TE-LTR and C-LTR cricoid cartilage vs. control cartilage and negative controls (muscle) shown with mean and 95% confidence intervals. Two high extreme outlier values of control cartilage and one of C-LTR is not shown as they are much higher on the y-axis. Figure 9B shows AP modulus vs. GAG content revealed reverse correlation which was statistically significant with (p=0.0407) with R-squared = 0.36. Figure 9C shows ML modulus vs. GAG content. This shows trend toward inverse correlation which is not statistically significant (p= 0.1536) and R-squared = 0.19.

Figures 1 OA- IOC provide images of the histology of 1% HA scaffolds after 12 weeks in vitro. Figure 10A shows Safranin O stain (5 Ox) revealing dense extra-cellular matrix. Figure 10B shows Safranin O stain (lOOx) showing the same as Fig. 10A at closer magnification. Figure IOC shows collagen II stain (50x) showing abundant high intensity staining. Figures 10D-10F provide images of the histology of 1% HA scaffolds after 12 weeks in vitro and 12 weeks in vivo. Figure 10D shows Safranin O stain (50x) of the extracellular matrix of cartilage. Figure 10E shows Von Kassa/Alcian Blue stain (50x) of the dense extracellular matrix. Figure 10 F shows collagen II stain (50x) showing dense staining for collagen II.

Figures 1 lA-1 ID shows images of bony HA construct after 12 weeks in vivo culture. Figures 11 A and 1 IB show Safranin O stain revealing decreased staining of extracellular matrix (50x). Figures 1 1C and 1 ID show Safranin O stain of different animal revealing ossification and lack of cartilage (lOOx).

Figures 12A-12C provide images of the histology of PGA constructs after 12 weeks in vitro. Figure 12A shows Safranin O stain revealing extracellular matrix (50x). Figure 12B shows Safranin O stain revealing decreased intensity of staining and remaining PGA strands (50x). Figure 12C shows collagen II immunostain revealing decreased staining intensity (50x). Figures 12D-12F provide images of the histology of PGA constructs after 12 weeks in vivo. Figure 12D shows Safranin O stain revealing decreased intensity of staining (5 Ox). Figure 12E shows Safranin O stain after 12 weeks in vivo revealing sparse center of the PGA construct (50x). Figure 12F shows collagen II immunostain revealing decreased intensity in the center (50x).

Figure 13 A provides a post-mortem view with Safranin O stain of 1% HA tissue- engineered cartilage graft (arrow) at 12 weeks post-op (20x). Figure 13B shows epithelial lining with Safranin O stain (50x). Figure 13C shows epithelial lining with Safranin O stain (lOOx). Figure 13D shows center of cartilage graft with Safranin O stain (lOOx). Figure 13E shows collagen II immunostain of construct revealing intense staining (50x). Figure 13F shows collagen I immunostain revealing lower intensity staining (50x).

Figure 13G shows collagen II immunostain of normal cricoid cartilage of the specimen revealing intense staining (50x). Figure 13H shows collagen I immunostain revealing minimal intensity staining (50x).

Figure 14A shows control LTR (20x) showing autologous thyroid graft at the top (arrow). Figure 14B shows epithelial lining with Safranin O stain (50x). Figure 14C shows center of cartilage graft (50x) revealing dense chondrocytes and matrix. Figure 14D shows transition zone (50x) revealing graft and fibrous tissue at junction. Figure 14E shows collagen II immune-stain (50x) revealing intense staining of the graft. Figure 14F shows collagen I immune-stain (50x) revealing no staining of the graft but some stain of the connective tissue.

DETAILED DESCRIPTION OF THE INVENTION

Herein, it is demonstrated for the first time that in vitro and in vivo derived tissue- engineered cartilage can be used for reconstructive surgery of the airway. The cartilage consistently survived 12 weeks in the larynx, become epithelized and acted as a diffracting graft with mechanical strength and histological properties similar to control cartilage. Indeed, thirteen TE animals with 1% HA as the scaffold material survived 12 weeks with evidence of an intact construct diffracting the airway and no inflammatory reaction with the HA material that is often found with other scaffold materials such as PGA or hyalograft C (Weidenbecher et al. (2007) Laryngoscope 117: 1745-9). These results show that HA in vitro and in vivo cultured, tissue-engineered cartilage constructs are an alternative to the grafts currently used and provide reliable cartilage with mechanical and histological properties similar to autologous cartilage. This novel approach combines both the controlled laboratory environment in vitro and the natural milieu of the body (e.g., neck) in vivo for maturing the cartilage. This is superior to the in vivo only approach in which there is no control of the tissue fate from the beginning. This process allows for the development of customizable cartilage for replacement therapy and LTR. Indeed, this technique has clinical application for the replacement of cartilage, including in the airway and head and neck region (e.g., auricular reconstruction for aural atresia or nasal reconstruction), due to trauma, disease, or cancer, or any other reason.

As explained in more detail hereinbelow in the Examples, 13 rabbits survived the tissue-engineered laryngotracheal reconstruction (TE-LTR) and there were only 2 deaths related to graft problems. One was due to the use of a polyglycolic acid (PGA) derived construct which caused a significant inflammatory response leading to animal's demise. There were un-degraded microfilaments of PGA after 6 months in the specimen and thus, no additional LTRs were performed with PGA grafts. The more rapid degradative properties of HA are well established (Chung et al. (2006) J. Biochem. Mat. Res., 77:518- 525; Zhao et al. (2014) J. Surg. Res., 187:394-402; Grayson et al. (2004) J. Biomaterial Sci. Polym., 15: 1281-04), while persistent synthetic scaffold material may elicit a significant inflammatory reaction (Weidenbecher et al. (2007) Laryngoscope 1 17: 1745-9; Sittinger et al. (1996) J. Biomed. Mater. Res., 33:57-63). Second, one animal died because a bony and brittle graft collapsed into the lumen of the airway resulting in airway obstruction. In this animal, all of the constructs had become bony after 12 weeks in vivo, and there were no other options for LTR. After this death bony grafts were avoided. Notably, the overall problem with bone formation was greatly reduced through the addition of the in vitro growth of the graft. The third HA death was unrelated to the laryngotracheal airway as an intact reconstruction was found on autopsy. The animal died of unclear reasons.

While the survival results presented in the initial study were not ideal, the LTRs in the rabbit model were riskier than LTR for humans, e.g., infants and children, for a number of reasons, including the smaller airway and, most importantly, the lack of overnight intensive care monitoring of the airway and support with stents or endotracheal tubes during the healing process. Since ventilation of the rabbit is not feasible, immediate extubation was chosen and worked in most cases. The few mortalities may have all been easily preventable with proper suctioning, intubation or endoscopic balloon dilation (Whigham et al. (2012) Ann. Otol. Rhinol. Laryngol, 121 :442- 448).

The mechanical testing of the TE-LTR, control LTR (C-LTR), and control cricoids offered interesting insight. On average, the numbers from the TE-LTR and C- LTR compression were comparable. Within each group, there was some variability which reflects inherent anatomical variability in animals. Furthermore, cricoid compression does not singularly test the construct, but instead tests the entire cricoid with the graft in position. This may introduce greater variability in the test. In contrast to anterior-to-posterior (AP) compression, which tests compression, the medial-to-lateral (ML) compression actually tests the tensile strength of the construct in the airway as it bends in situ. More localized material property testing could be performed with nano- indentation devices, which employ a microelectronic sensor cantilever device. This testing can measure the surface microscopic mechanical properties of just the construct itself (Moyer et al. (2012) J. Biomech., 45:2230-5).

In general, 1% HA out-performed PGA alginate as a scaffold matrix. The mechanical properties of the 1% HA constructs more closely resembled that of autologous cartilage than did the PGA constructs after 12 weeks in vitro and after 12 weeks in vivo. The PGA constructs were softer after 12 weeks in vitro and did not reach the mechanical strength of HA constructs after an additional 12 weeks in vivo. After 12 weeks in vitro followed by 12 weeks in vivo, the HA grafts on average retained the same flexural moduli as calculated after 12 weeks in vitro, while the PGA grafts' moduli increased more dramatically after the 12 weeks in vivo period. Without being bound by theory, the HA constructs mature earlier than PGA constructs. The HA constructs had the opportunity to reach nearly full mechanical potential in vitro, taking more advantage of a controlled environment. There was also dramatic increase in the thickness (and volume) of the all constructs after the in vitro incubation.

The addition of the in vitro arm with addition of TGF- 3 locked in the cartilage morphology early although bone later developed in varying degrees in the constructs of some animals. Osteocytes and chondrocytes originate from the same precursor mesenchymal stem cells (Schraufstatter et al. (201 1) Front. Biosci., 16:2271-88). It is possible that the constructs that ultimately produced bone in vivo, were incompletely differentiated in vitro, and the in vivo culture directed the cells to form into osteocytes. Accordingly, more complete differentiation (e.g., by longer incubation) in vitro would correct this possibility.

GAG is an essential macromolecule for the proper function of hyaline cartilage, possibly related to the negative charge of the GAG side chains which attracts and retains water molecules aiding in the compressibility of cartilage which is necessary for weight- bearing joints such as the knee (O'Connell et al. (2014) Eur. Cell Mater., 27:312-20; Asanbaeva et al. (2007) Arthritis Rheum., 56: 188-98). However, it is unclear why GAG would be important for a rigid supporting graft in the airway. In fact, there was a reverse correlation ultimately between GAG content of the HA constructs used in the LTR and the AP compressive modulus suggesting GAG may not be an important component for LTR graft stiffness (strength) and functionality, as collagen content and fibrosis are likely more relevant and may in fact increase as GAG decreases (Wilusz et al. (2014) J. Mech. Behav. Mater., 38: 183-97; Guilak et al. (1999) Osteoarthritis Cartilage 7:59-70).

In terms of histology, it is clear that both the HA and PGA constructs generated cartilaginous tissue, but that the HA constructs were more homogenous and more closely resemble the histological appearance of normal cartilage compared to the PGA derived constructs. The cellularity of the PGA constructs was generally more sparse than that of the HA specimens. The finding of the lower density of chondrocytes in the center portion of the constructs has a number of possible explanations including limited oxygen or nutrient diffusion through PGA which could lead to cellular death and reduced viability (Ellis et al. (2001) Magn. Reson. Med., 46:819-26). The PGA appeared more susceptible to this problem. A second possible explanation is the accumulation of lactic acidic, a specific breakdown product of PGA, could lower the local pH to levels which are toxic to chondrocytes in the construct center (Bujia et al. (1995) Laryngorhinootologie 74: 183-7). The use of thinner constructs can improve diffusion to the central regions and augment cellular viability. In addition, hydrogels may be used to supplement particle transport and homogenize the grafts (Vladescu et al. (2012) J. Pharm. Sci., 101 :436-42). Moreover, synthetic hollow fibers may be used to delivery nutrients and compounds to the center of the construct (Bettahalli et al. (2011) Acta Biomater., 7:3312-24; Ellis et al. (2001) Magn. Reson. Med., 46:819-26).

Protection from bone formation may aided by placement of the constructs deep into the musculature rather than superficially. This would protect the constructs from dermal fibroblast infiltration and dedifferentiation into bone formation either caused by contamination in the animal or de-differentiation in vivo. The tissue properties are shown to "remodel" (histologically and mechanically) during the 12 weeks in vivo. Over the long-term, the milieu of the in vivo environment including cells, cytokines and other exocrine signals influence tissue fate. Changes in cartilage even occur with free autologous cartilage implanted in the airway over time (Jacobs et al. (1999) Ann. Otol. Laryngol. Rhinol, 108:599-605).

In accordance with the instant invention, methods of synthesizing cartilage are provided. The methods comprise first culturing chondrocytes in vitro to form a cartilage construct and then culturing the cartilage construct in vivo. The methods may further comprise obtaining the chondrocytes from a subject. The methods may further comprise harvesting or isolating the cartilage construct (e.g., insert) from the host after the in vivo culturing step. The isolated cartilage construct may then be used to replace cartilage in a subject. The isolated cartilage may be shaped for the intended purpose prior to insertion into the subject for therapeutic treatment. The isolated cartilage may be maintained in a composition comprising a pharmaceutically acceptable carrier.

The in vitro culturing step of the instant invention comprises culturing

chondrocytes under conditions which promotes chondrocyte growth and the formation of cartilage. The chondrocytes used for in vitro culturing may be individual cells, a sheet of chondrocytes, or a piece of cartilage comprising chondrocytes. The chondrocytes may be dissociated prior to culturing. In a particular embodiment, the chondrocytes are maintained in media comprising at least one chondrogenesis factor. A chondrogenesis factor may be a growth factor or cytokine which promotes the growth of chondrocytes and formation of cartilage, particularly to the exclusion of osteoclasts or bone.

Chondrogenesis factors include, without limitation, transforming growth factor (TGF)- (e.g., TGF-βΙ, TGF- 2, or TGF- 3), insulin-like growth factor (IGF) (e.g., IGF-I or IGF- II), fibroblast growth factor (FGF) (e.g., basic fibroblast growth factor (bFGF; FGF-2), FGF-5, or FGF- 18), vascular endothelial growth factor (VEGF), and bone morphogenetic proteins (BMP) (e.g., BMP-2, BMP-4, or BMP-6). In a particular embodiment, the chondrogenesis factor is TGF-β, particularly TGF- 3. The chondrocytes may be maintained under conditions which promotes chondrocyte growth until the formation of a cartilage construct. The conditions may be static or may involve movement (e.g., rotating and/or shaking incubator). In a particular embodiment, the chondrocytes are maintained in a media comprising at least one chondrogenesis factor for at least one week, particularly at least four weeks, at least eight weeks, or at least 12 weeks. In a particular embodiment, the chondrocytes are maintained in a media comprising at least one chondrogenesis factor for about eight to about 36 weeks, particularly about ten to about 24 weeks, about ten to about sixteen weeks, about twelve to about sixteen weeks, or about 12 weeks. In a particular embodiment, the in vitro culturing is for at least about 12 weeks.

In addition to the chondrogenesis factor, the chondrocytes may also be cultured in vitro with a scaffold. The scaffold may comprise a biocompatible material, particularly a material that is not antigenic. In a particular embodiment, the scaffold comprises hyaluronic acid. In a particular embodiment, the hyaluronic acid is present at an amount from about 0.1% to about 10%, about 0.1% to about 5%, about 0.1% to about 3%, about 0.25% to about 2%, or about 0.5% to about 1.5%. In a particular embodiment, the culture conditions comprise about 1% hyaluronic acid (e.g., w/w). In a particular embodiment, the hyaluronic acid is polymerized (e.g., ultraviolet photopolymerized). The

chondrocytes may be cultured in vitro in a scaffold of a desired shape to direct the growth and formation of the cartilage construct.

Chondrocytes for use in the methods of the instant invention may be obtained from cartilage. In a particular embodiment, the chondrocytes are obtained from an animal, particularly a mammal, more particularly a human. The chondrocytes may be obtained from the subject to be treated with the synthesized cartilage (i.e., they are autologous). Chondrocytes may be obtained from any cartilage in a subject. For example, the chondrocytes may be obtained from auricular, larynogeal, costal, or nasal (e.g., alar or septal) cartilage.

After the in vitro culturing, the cartilage construct is further cultured in vivo. The cartilage construct may be cultured within any animal, particularly a mammal, more particularly a human. In a particular embodiment, the cartilage construct is cultured in the subject to be treated with the final engineered cartilage. The in vitro cultured cartilage construct may be inserted into any feasible location of the subject. In a particular embodiment, the cartilage construct is inserted subcutaneously (e.g., into a subcutaneous pocket). For example, the cartilage construct may be inserted into the neck (e.g., subplatysmal). The cartilage construct may also be inserted intramuscularly, e.g., into the muscle of the subject (e.g., into the neck muscles). In a particular embodiment, the cartilage construct is inserted into the chest. In a particular embodiment, the cartilage construct may be inserted into the subject where the original chondrocytes/cartilage was harvested (e.g., at or near (e.g., same region or body part (e.g., ear)) the site of extraction). In a particular embodiment, the cartilage construct may be inserted into the subject where the final cartilage after in vivo culturing will be implanted (e.g., at or near (e.g., same region or body part) the site of implant). In a particular embodiment, the cartilage construct is maintained with/within a scaffold (as described above for in vitro methods) when cultured in vivo. The cartilage construct is maintained in vivo for maturation of the cartilage. In a particular embodiment, the cartilage construct is maintained in vivo for at least one week, particularly at least four weeks, at least eight weeks, or at least 12 weeks. In a particular embodiment, the cartilage construct is maintained in vivo for about eight to about 36 weeks, particularly about ten to about 24 weeks, about ten to about sixteen weeks, about twelve to about sixteen weeks, or about 12 weeks. In a particular embodiment, the in vivo culturing is for at least about 12 weeks. The in vitro and in vivo culture times may be the same or different. In a particular embodiment, the in vivo culture time is greater than the in vitro culture time.

The instant invention also encompasses methods of treating a subject in need of cartilage repair and/or replacement. The subject may be a child or an adult. The method comprises inserting the cartilage synthesized by the methods of the instant invention into the subject where needed. In a particular embodiment, the method comprises

synthesizing the cartilage by the methods of the instant invention and then inserting the cartilage into the subject in need thereof. The cartilage synthesized by the methods of the instant invention may be used in orthopedic applications. The cartilage synthesized by the methods of the instant invention may be used to replace or repair any cartilage in the body including, without limitation, cartilage of the ear, cartilage of the nose, cartilage of joints (e.g., cartilage of the knee (articular or meniscus), ankle, shoulder, etc.), cartilage of the hip, cartilage of the shoulder, cartilage of the rib cage, cartilage of the larynx, cartilage of the trachea, and cartilage between vertebral discs. The need to replace or repair cartilage in a subject can be due to any disease or disorder (e.g., a degenerative disorder; a congenital defect), traumatic injury (e.g., a tear or loss of cartilage), or cancer. The methods of the instant invention may be used to reconstruct areas of cartilage (i.e., reconstructive surgery) including, without limitation, reconstructing the nose or ear.

Methods of inserting cartilage are well known in the art. As an example, an incision may be made near the damaged or deficient cartilage. An amount of healthy and/or damaged cartilage and/or surrounding bone may be removed, if necessary, for proper fitting of the engineered cartilage. The engineered cartilage may be modeled, carved, cut, or modified prior to insertion. The engineered cartilage may be slightly smaller or slightly larger (e.g., from about 0.1% to about 10% smaller or larger) than the site for insertion. The inserted engineered cartilage may also be adhered to bone, as needed, by methods known in the art. The incision may then be sutured by any known method. Definitions

The following definitions are provided to facilitate an understanding of the present invention:

The singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, the terms "host," "subject," and "patient" refer to any animal, particularly mammals including humans.

"Pharmaceutically acceptable" indicates approval by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

A "carrier" refers to, for example, a diluent, adjuvant, preservative (e.g.,

Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid, sodium metabisulfite), solubilizer (e.g., Tween® 80, polysorbate 80), emulsifier, buffer (e.g., Tris HC1, acetate, phosphate), antimicrobial, bulking substance (e.g., lactose, mannitol), excipient, auxiliary agent or vehicle with which an active agent of the present invention is administered. Pharmaceutically acceptable carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin. Water or aqueous saline solutions and aqueous dextrose and glycerol solutions may be employed as carriers, particularly for injectable solutions. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin (Mack Publishing Co., Easton, PA); Gennaro, A. R., Remington: The Science and Practice of Pharmacy, (Lippincott, Williams and Wilkins); Liberman, et al, Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y.; and Kibbe, et al, Eds., Handbook of Pharmaceutical

Excipients, American Pharmaceutical Association, Washington.

The term "treat" as used herein refers to any type of treatment that imparts a benefit to a patient afflicted with a disease, including improvement in the condition of the patient (e.g., in one or more symptoms), delay in the progression of the condition, etc.

The following example is provided to illustrate various embodiments of the present invention. The example is illustrative and is not intended to limit the invention in any way.

EXAMPLE MATERIALS AND METHODS

Animal Care

All procedures, animal care and housing were approved by the Institutional Animal Care and Use Committee (IACUC). All procedures were in full compliance with the IACUC protocol and National Institute of Health guidelines.

Autologous Chondrocyte Harvest and Isolation

Under intravenous and inhalational general anesthesia and sterile conditions, a 4x4 mm portion of auricular cartilage was harvested from New Zealand white rabbits (3- 3.5 kg). The cartilage was minced and digested overnight in 0.1% collagenase II (w/v). The isolated cells were cultured and expanded in chondrocyte growth media (CGM) (Lonza) in a humidified 5% CO₂, 37°C incubator until sufficient number of cells were available for the generation of cartilage constructs.

Generation of Tissue-Engineered Cartilage Constructs

Chondrocytes were detached before confluence with 0.25% trypsin-EDTA and seeded at 50xl0⁶ cells/ml into 1 of 2 scaffold materials: photocrosslinked,

methylacrylated 1% HA or calcium alginate encapsulated PGA mesh. For the former, the cells were suspended in HA, cast in a mold, UV cross-linked and cut to size (Kim et al. (2011) Biomaterials 32:8771-82). For the latter, cells were suspended in 1.2% sodium alginate, seeded onto pre-cut pieces of PGA mesh (Synethcon) and polymerized by suspension in 102 mm calcium chloride for 5 minutes (Yang et al. (2013) ACS Appl. Mater. Interfaces, 5: 10418-22). The engineered constructs were cultured in defined chondrocyte differentiation media (CDM; Life Sciences/Sigma), of which the main cytokine was TFG- 3 at lOng/ml (Bian et al. (201 1) Biomaterials 32:6425-34; Kim et al. (2012) J. Mech. Behav. Biomed. Mater., 1 1 :92-101). The media also contains

Dulbecco's modified Eagle medium (DMEM), supplemented with 0.1 uM

dexamethasone, 50 ug/ml ascorbic acid, 40 ug/ml prolene, InM sodium pyruvate, 6.25 ug/ml insulin, 6.25 ug/ml transferring, 6.25 ng/ml selenous acid, 1.25 mg/ml bovine serum albumin, 5.35 ug/ml linoleic acid, 100 U/ml penicillin, 100 ug/ml streptomycin, and 2.5 ug/ml fungizone.

Optimization of In Vitro Time, Culture Conditions and Scaffold Composition

In order to determine the optimal scaffold material and length of in vitro culture time, chondrocytes were seeded at a concentration of 50 million cells/ml into 1 of 3 materials (1%, 2% HA w/v or PGA-calcium alginate and cut into cylindrical disks (4 mm diameter and 4 mm height) (Figure 2A). The discs were cultured in either static culture (floating in CDM) or in a rotating wall vessel bioreactor (Rotary Cell Culture systems, Synthecon, Inc.) with CDM. They were placed in a humidified 5% CO₂, 37°C incubator for 4, 8 or 12 weeks. Four constructs of each condition were taken out at 4, 8 or 12 weeks, and 3 underwent compression testing (Figure 2A) and 1 underwent histological analysis and GAG content determination.

In Vitro Culture of Tissue-Engineered Cartilage Constructs

Based on the optimization results, chondrocytes were cultured in vitro for 12 weeks in either 1% HA or PGA-alginate. First, for each animal 7 cross-linked 1% HA constructs were cut to the dimension of 13 mm (length) x 5 mm (width) x 2.25 mm (thickness) as an estimated size of an LTR graft. Seven PGA constructs were cut to the same length and width with a manufactured thickness of 2 mm. The constructs were then cultured on a shaking platform which was chosen over static culture based upon the optimization results and was chosen over the bioreactor because of the concern for large construct collisions. After 12 weeks of culture, 4 HA and 4 PGA constructs were randomly selected for neck implantation, and one of each type was selected for mechanical testing and one for histological and biochemical analysis. Both specimens were photographed, weighed, and measured in all dimensions.

Implantation of the Constructs

Under general anesthesia, 4 HA and 4 PGA tissue-engineered constructs were implanted into the left and right side of the neck, respectively, of the corresponding rabbit. A 5-6 cm transverse incision was made slightly below the cricoid, and a subplatysmal pocket was developed in the neck. The constructs were placed inside the subplatysmal pockets and the pockets were sutured close. The implantation sites pockets were tagged with permanent, colored sutures to facilitate future harvesting.

Harvesting of Constructs and Tissue-Engineered Laryngotracheal Reconstruction (TE- LTR)

The constructs were harvested under general anesthesia 12 weeks after implantation. A transverse incision was made slightly below the cricoid. The neck was explored in the sub-platysmal plane, and all constructs were isolated and extracted (Figures 4B). The most optimal construct was selected as a graft for TE-LTR based on gross appearance of adequate length (>13 mm), width (5 mm) thickness (2.5 mm) and minimal or no bone formation (Figures 4B, 4C). The cricoid and the first 2 tracheal rings were divided in the mid-line. The graft was sutured between the cricoid plates to diffract and expand the subglottic airway (Figures 5A, 5C). An air-leak test was performed with saline and positive pressure in order to ensure that the graft was secure. The wound was closed in layers, and the rabbit was monitored for signs of respiratory distress. The remaining constructs were photographed, weighed, measured and underwent mechanical testing and histological and biochemical analysis.

Control LTR: The same surgical procedure described above was used for the control LTR (C-LTR; Figure 5E). However, instead of a tissue-engineered construct, autologous thyroid alar cartilage was harvested, sculpted to 13x5 mm, and used as a graft to expand the airway.

Harvest of Reconstructed Larynx and Endoscopic Examination

The TE-LTR (n=13) and C-LTR (n=18) animals were humanely euthanized after 12 weeks according to the approved IACUC protocol. The larynges were excised with a portion of trachea. The airway underwent endoscopic examination with a 4 mm, zero and 30 degree Storz-Hopkins Telescope (Karl Storz Inc.; Figures 5B, 5D, 5F). Still and video images were recorded and stored on secure encrypted devices. The larynx and cricoid including the LTR graft were then isolated for mechanical and histological testing. In addition, a small portion of the tissue-engineered and control (thyroid) cartilage underwent biochemical analysis for GAG content determination as outlined below.

Thirteen control animals were euthanized, and their larynges were harvested. All prematurely deceased animals underwent autopsy with dissection of the laryngotracheal complex. The reconstructed larynx was photographed and underwent histological analysis, but was not mechanically tested.

Mechanical Testing of the In Vitro and In Vivo Constructs

The constructs underwent mechanical testing for flexural modulus after twelve weeks in vitro and then after twelve weeks in vivo using a three-point bend test. A rectangular three-point bending technique (Mauck, R. Penn Center for Musculoskeletal Disorders, Biomechanics Core. Rectangular specimen in three point bending test.

www.med.upenn.edu/pcmd/BCTestingMethods.shtml) was employed to test the mechanical properties of the cartilage grafts. The specimens were placed on two supports, 8 mm apart within an Instron 5542 (Norwood, Mass) uniaxial testing frame (Figure 2C). The actuator applied constant downwards point deformation at the midpoint of the specimen at a constant rate of 1 mm/min. Reaction forces and displacements were measured as functions of time throughout the test at a rate of 100 Hz. The Instron records time, extension (displacement), and load (force). Tests were stopped at 3 mm of deformation or when failure occurred. Forces and displacements were normalized to stresses and strains based upon the geometry of each specimen. The height (h), base (b), and length (1) of the specimens were determined using image analysis software (ImageJ, NIH). The flexural modulus was calculated by determining the slope of a line that best fit the linear portion of the stress-strain curve generated. Results were compared to autologous thyroid cartilage of the same dimensions. Mechanical Testing of TE-LTR, Control-LTR and Control Cricoids

Both anterior-to-posterior (AP) and medial-to-lateral (ML) compression were performed on the isolated cricoid with the construct intact. The compression test was applied to the cricoid with two parallel plates, using the same actuator as above with constant deformation of 1 mm/min (Figure 2B). The modulus of each specimen was calculated by fitting a line to the slope of stress-strain curve generated. The AP and ML compressive moduli of each TE-LTR cricoid were compared to C-LTR and to control cricoids. Histological, Immuno-Histochemical and Biochemical Analysis

One HA and PGA construct from each animal after both in vitro and in vivo incubation was used for histology, immunohistochemistry and biochemical analysis. The specimens were fixed in 10% neutral-buffered formalin for at least four days and were embedded in parafilm and underwent sectioning and staining with hemotoxylin & eosin, Safranin O and alcian blue. In addition, sections were immunostained-stained for collagen Types I and II (Kim et al. (2011) Biomaterials 32:8771-82). In addition, a small portion of the construct was excised and underwent a GAG content determination which is measured as percentage weigh/total weight (% w/w). The net weight of the entire construct was measured after digestion for 16 hours with papain (1 ml/sample), 0.56U/ml in 0.1 M sodium acetate, 10 M cysteine hydrochloric acid, and 0.05 M

ethylenediaminetetraacetic acid (pH 6). GAG content (w/w) was determined using 1, 9 dimethyl-methylene blue assay (Kim et al. (2011) Biomaterials 32:8771-82; Bian et al. (2011) Biomaterials 32:6425-34).

After mechanical testing, all the cricoid specimens (TE-LTR, C-LTR and cricoid controls) were fixed in 10% neutral-buffered formalin for at least 4 days and then decalcified in formic acid for an additional three days. Samples were then histo- processed to paraffin, embedded and sectioned at 4 um. Slides were stained with hematoxylin and eosin (nuclei stains blue and cytoplasm red) (Survarna et al, The hematoxylins and eosin. In: Survarna SK, Layton C, Bancroft JD, editors. Theory and practice of histological techniques, 7th ed. Philadelphia, PA: Churchill- Livingstone/Els evier, 2012, p.173-186), and Safranin O (cartilage mucin, mast cell granules stain red, nuclei black, and cytoplasm blue). Some were stained with Von Kossa/Alcian Blue (cartilage stains blue, nuclei stains pink, and bone stains black) (Survarna et al, Connective and mesenchymal tissues with their stains. In: Survarna SK, Layton C, Bancroft JD, editors. Theory and practice of histological techniques, 7th ed.

Philadelphia, PA: Churchill-Livingstone/Elsevier, 2012, p.187-214), and some underwent type II Collagen immunohistochemistry. Collagen II, which is a main component of cartilage, stains brown with blue nuclei (Roberts et al. (2009) Knee 16:398-405). GAG content of a small portion of the construct was quantitatively evaluated (Kim et al. (201 1) Biomaterials 32:8771-82; Bian et al. (2011) Biomaterials 32:6425-34).

Statistical Analysis

For change in construct thickness, all HA and PGA constructs started with a thickness of 2.25 and 2.00 respectively. The mean thickness of HA and PGA was calculated with SD and 95% confidence intervals. The optimization study was evaluated using one-way ANOVA with Tukey's post hoc analysis to determine statistical differences between groups. The data for the in vitro, in vivo and cricoid mechanical testing as well as the GAG content were determined to violate suppositions of normality. With more than 2 in all groups, the comparisons were made using non-parametric Kuskall-Wallis (non-parametric version of ANOVA) tests of statistical significance with post hoc pairwise analysis using Bonferroni correction. In addition, a linear regression analysis was performed for GAG content versus AP and ML compressive modulus for each animal and R coefficient was determined. In all cases statistical significance was indicated by p<0.05.

RESULTS

Viable tissue-engineered cartilage was produced in an immunodeficient mouse model. Human chondrocytes in chondrocyte growth media (CGM) were seeded on 5x3 mm PLA scaffolds (OPLA, BD Sciences). The effect of engraftment techniques were studied comparing in vitro tissue-engineered constructs in static culture to in vivo subcutaneous implanted constructs. In addition, the effect of calcium alginate encapsulation was compared to no alginate. After 3 and 6 months, the constructs were harvested for gross and histological examination. All constructs demonstrated viable cells. However, only the alginate encapsulated constructs cultured in vivo demonstrated mature cartilage. The 6-month constructs were qualitatively more optimal than the 3- month constructs. The 6-month constructs demonstrated highly cellular cartilage with robust extracellular matrix on hemotoxylin and eosin staining. Moreover, the constructs in the mice were too small to undergo mechanical testing and therefore only used for qualitative histology.

The first approach to tissue-engineered rabbit model was to employ an entirely in vivo protocol in which autologous cartilage tissue was harvested from each rabbit, the cells were isolated and expanded, and then the chondrocytes were seeded in either hyaluronic acid (HA) or polyglycolic acid (PGA)-alginate scaffolds. The constructs were then implanted in vivo for 24 weeks, and then an LTR was performed. After an additional 12 weeks, the animals were sacrificed for histological and mechanical testing of the cricoid. The 24-week in vivo only period exposed the constructs to an environment that lacked control and predictability, and ultimately led to unsatisfactory results. While some cartilaginous tissue was generated using this approach, most of the constructs became consistently and predominantly ossified in vivo. Both osteocytes and chondrocytes originate from mesenchymal stem cells (Schraufstatter et al. (2011) Front. Biosci., 16:2271-88). It was therefore postulated that the constructs were either incompletely differentiated and the in vivo environment favored the formation of osteocytes, or that the cells became dedifferentiated and then re-differentiated into osteocytes in vivo (Astachov et al. (201 1) Front. Biosci., 16:261-76).

It was hypothesized that introducing an in vitro culture period before

transplantation in vivo would provide a more controlled environment, in which the tissue- engineered cartilage could more effectively evolve and differentiate (Doolin et al. (2002) J. Ped. Surg., 37: 1034-1037). It was theorized that this strategy would "lock in" cartilaginous properties prior to subsequent implantation in vivo for an additional time period of 12 weeks, which was intended to explore for any additional change and maturation in cartilage morphology. Autologous chondrocytes, harvested from the ear, were suspended in either hyaluronic acid (HA) or polyglycolic acid (PGA) in order to generate constructs in vitro before placing them back into the subcutaneous tissue of the animal's neck. The in vitro time course, which was determined by the optimization section of the study, precedes an additional in vivo time period of 12 weeks. Seven of the grafts were harvested for histological and mechanical testing, and one HA graft (in one case, a PGA graft) was selected for use in laryngotracheal reconstruction (LTR)(Figure 1).

To elucidate the overall feasibility of the TE technique, 12 weeks after LTR, the complete reconstructed airway underwent endoscopy, photography, GAG content determination, histological analysis and mechanical testing. The results from the tissue- engineered specimens were compared to two controls: 1) intact cricoids and 2) cricoids reconstructed with autologous thyroid cartilage. It was determined whether in vitro and in vivo cultured, tissue-engineered cartilage constructs sufficiently emulate the histological and mechanical properties of autologous cartilage and whether these constructs are effective as expansion grafts for LTR. Optimization of the Scaffold Composition and In-Vitro Culture Time and Bioreactor Conditions

The modulus was the highest for the 1% HA static constructs cultured in static culture for 12 weeks (mean=2.99 MPa, SD=0.39) in vitro, closely followed closely by the 1% HA (mean=2.38, SD=0.47) cultured in the rotary bioreactor. The 1% HA static had a higher modulus at 12 weeks compared to the 2% HA (p= 0.0016) and PGA (p=0.0003). The GAG content was also highest in the 1% HA bioreactor specimens, abeit with only one specimen at each point in the study.

Gross and Endoscopic Analysis

Figure 5 displays three representative sets of images of the airway surgery, depicting the larynx immediately after the HA construct had been sutured in place (Figures 5A, 5C) or after the C-LTR (Figure 5E) and on post-mortem endoscopy 12 weeks post-operative (Figure 5B, 5D, 5F). At 12 weeks post-operative, the HA constructs in the surviving animals were well integrated into the airway, similar to the C- LTR.

Survival after Airway Surgery

Thirteen out of the 16 rabbits that underwent TE-LTR survived. One rabbit died due to a large inflammatory granulation reaction to the PGA (synthetic polymer) material (Figures 6A-6D). Because of that result, PGA-derived scaffolds were not used for LTR. A second rabbit died due to the collapse of a brittle bony graft in the airway on postoperative day ten. When attempting to suture the graft between the cricoid plates, it was obvious that the construct was abnormally brittle and inflexible (Figures 6E-6G).

Histological analysis confirmed that the graft was in fact bony (Figures 6G, 6H) which led to the premature demise. A third animal died suddenly on post-operative day four for no discernible cause on autopsy. The construct appeared intact in the airway, although it was inset at a slightly lower profile due to technical error. However, it did not obstruct the airway. Overall, HA constructs supported normal breathing and activity for 12 weeks in 13 out of 15 rabbits. Nineteen of the 20 C-LTR animals survived, but one animal had a severe prolonged wound infection and was excluded from the final analysis.

Mechanical Analysis of Constructs in vitro and in vivo After 12 weeks in vitro, HA constructs (n=18; Figure 4B) had a mean flexural modulus of 1.21 Mpa (SD=0.53), compared to the PGA (n=9) constructs which had an mean flexural modulus of 0.43 MPa (SD=0.31 ; p=0.0024; Figure 7A). The mean modulus after an additional 12 weeks in vivo for HA constructs (n=19) was 1.55 MPa (SD=2.37) and for PGA constructs (n=17) was 0.41 MPa (SD=0.27; ns, p=0.097; Figure 7B). The average modulus for HA construct increased by 0.34 MPa (28%) during the in vitro culture while, the average modulus of the PGA constructs decreased slightly by 0.02 MPa (5%; Figures 7C, 7D), but neither difference was significantly different (p=0.8160 and p=0.9999, respectively). The mean modulus of the thyroid control cartilage (n=12) was 9.76 MPa (SD=8.09) compared to the mean modulus of 1.55 Mpa for the HA in vivo constructs (p=0.0054).

Compression Testing of TE-LTR, C-LTR and Control Cricoids

The cricoid controls (n=13), which had not undergone any surgical procedures, were used to evaluate the compressive modulus of the natural airway. The mean AP modulus and ML modulus of the native cricoids was l.38 MPa (SD=0.53) and 2.34 MPa (SD=1.24), respectively. The TE-LTR (n=13) had an average AP and ML modulus of 0.75 MPa (SD=0.40) and 1.34 (MPa (SD=0.82), respectively, while the C-LTR larynges had a mean AP and ML modulus of 1.16 MPa (SD=0.62) and 1.36 MPa (SD=1.00) respectively (Figures 8A, 8B). The control cricoids revealed higher AP and ML modulus compared to the TE-LTR (p=0.004, 0.047, respectively). Specifically the TE-LTR AP modulus was 54% of the native control cricoids (p=0.0369), while the ML modulus was 57%. There were no statistically significant differences between the TE-LTR and C-LTR for AP modulus (p =0.11 1) or ML modulus (p= 0.999). Overall the mean ML moduli and variability (SD) were higher than the AP moduli (Figures 8B, 8D).

GAG Content Analysis

No statistical difference was seen in GAG content between the HA constructs (n=l l, mean=2.57%w/w, SD=1.84) and PGA constructs (n=7, mean=1.78w/w, SD=1.14) after in vitro culture (p =0.9999; Figure 9A). Similarly, after in vivo implantation, no statistical difference (p=0.9999) was seen in GAG content between HA (n=25, mean 1.76% w/w, SD=1.27) and PGA constructs (n=20, mean 2.66% w/w, SD=2.41). The mean GAG of HA constructs after in vivo culture was less than the control cartilage (n=19, mean 7.50% w/w, SD=6.30, p=0.0001) and more than the negative control of muscle (n=8, mean 0.18% w/w, SD=0.04, p=0.0004). The HA constructs on average lost 32% of their GAG content during the 12 weeks in vivo, while PGA increased by 49%. The differences between HA in vitro and in vivo and between PGA in vitro and in vivo was not statistically significant (p=0.9999 for both; Figures 9B, 9C).

Cricoids

The TE-LTRs (n=12) averaged 2.55% w/w (SD=2.22), while the C-LTR (n=15) averaged 3.57% w/w (SD=3.29) compared to the control cartilage (n=19, mean 7.50% w/w, SD=6.30) and the muscle (0.18% w/w, SD=0.04) (Figure 10A). There was no statistically significant difference between the TE-LTR and C-LTR groups (p=0.999). There were statistically significant differences between the TE-LTR constructs, the control cartilage group (p=0.0028), and the control muscle (p=0.0010). The HA constructs, after 12 weeks in situ in the airway, had a lower GAG content than expected. However, it is important to note that this is also true for the C-LTRs. Figures IOC and 10D reveal a reverse correlation between the AP modulus and GAG content (R=0.36, p=0.0407) and a trend toward a reverse correlation between ML modulus and GAG content (R= 0.19, p=0.1536), which is not statistically significant.

Gross and Histological Analysis

All the HA and PGA constructs were cartilaginous on gross and microscopic examination after the 12 weeks in vitro. Twenty -three out of 37 HA constructs were cartilaginous after in vivo incubation whereas 17 out of 24 PGA constructs were cartilaginous (p=0.586 by Fisher's Exact Test). The constructs dramatically increased in thickness (and volume) during the 12 week in vitro culture. The HA constructs increased from 2.25 mm in thickness at manufacture to an average of 3.5 mm after in vitro incubation (56% increase, SD=0.4 SD; 95% CL4.3-2.7) and the PGA increased from approximately 2.0 mm in thickness to an average of 4.2 mm after in vitro incubation (120% increase, SD=0.4, 95% CI: 5.0-3.4). Small decreases in thickness were seen after the 12 weeks in vivo incubation (HA mean=3.9 mm, SD= 0.4, 95% CI: 4.7- 3.1); PGA (mean=3.9 mm, SD=0.4, 95%, CI: 4.9-2.9). From histology, it is clear that both the HA and PGA constructs generate cartilaginous tissue, but that the HA grafts are more homogenous and more closely resemble the histological testing of autologous cartilage (based on staining, cellular content, and quantitative GAG content). After 12 weeks in vitro, the HA constructs contained numerous chondrocytes with dense surrounding extracellular matrix (Figures 1 1A, 1 IB). Collagen was demonstrated on type II collagen stains (Figure 1 1C), with minimal staining on type I collagen. After 12 weeks in vivo, the HA constructs appeared to be producing an equivalent amount of extracellular matrix (Figure 1 ID, 1 IE), and more collagen (Figure 1 IF). Many of the constructs had become encapsulated and the chondrocytes still appeared viable. It is important to note that 14 out of the 37 in vivo HA constructs had at least some degree of bone formation which was worse in some animals than others. A few constructs, after 12 weeks in vivo, were predominantly bony rather than cartilaginous (Figure 12).

PGA constructs were consistently less satisfactory than the HA constructs. After 12 weeks in vitro, the PGA constructs produced less extracellular matrix (Figures 13A, 13B), and less collagen (Figure 13C). The chondrocytes also appeared smaller and less compact (Figures 13 A, 13B). In vivo, these differences persisted. The extracellular matrix seemed to decrease (Figures 13D, 13E) and the collagen II seems less abundant (Figure 13F). Most importantly, the cells in the center of the PGA constructs were extremely sparse after 12 weeks in vivo (Figures 13D, 13E), indicating that many of the cells on the inside of the PGA constructs did not survive. Further, as illustrated in Figures 6C and 6D, there are still abundant, large strands of residual PGA filaments in the construct after 12 weeks in vivo, but there is no evidence of residual HA in those in vivo constructs. Negative, inflammatory effects were seen with the granulation formation in the deceased animal that underwent LTR with the PGA construct (Figures 6A, 6B).

Histological Analysis of the LTR Specimens

Figure 14 displays representative images of one TE-LTR and Figure 15 displays one C-LTR. Both images demonstrated intact viable cartilage and fully epithelialized airway. Evidence of histologically viable cartilage was noted in 1 1 out of 13 surviving LTR constructs with only 1 having predominantly bone. In one animal, the construct was replaced by mostly fibrous tissue, but the airway remained satisfactory diffracted.

Overall, the cellular content of HA constructs after 12 weeks postoperative was similar to that of the control LTRs. The HA constructs were solidly integrated into the airway and were covered with a ciliated respiratory epithelium. There was no evidence of an intraluminal inflammatory reaction in any surviving animal. In addition, the HA constructs supported normal breathing and activity in 13 out of 15 specimens. Finally, the biomaterial was technically easy to suture, similar to normal hyaline cartilage (Figures 4C, 4D). While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it is not intended that the invention be limited to such embodiments. Various modifications may be made thereto without departing from the scope and spirit of the present invention, as set forth in the following claims.

Claims

WHAT IS CLAIMED IS

1. A method of synthesizing cartilage, said method comprising

a) culturing chondrocytes in vitro in the presence of at least one chondrogenesis factor for sufficient time to synthesize a cartilage construct; and

b) culturing the cartilage construct obtained in step a) in an animal host.

2. The method of claim I, further comprising isolating the cartilage construct obtained in step b).

3. The method of claim I , wherein said animal is a human.

4. The method of claim I, wherein said chondrogenesis factor is transforming growth factor β (TGF-β).

5. The method of claim 4, wherein said chondrogenesis factor is TGF- 3.

6. The method of claim I, wherein step a) comprises culturing said chondrocytes with a scaffold.

7. The method of claim 6, wherein said scaffold comprises hyaluronic acid.

8. The method of claim 7, wherein said scaffold comprises about 1% hyaluronic acid.

9. The method of claim I , wherein said in vitro culturing is for at least about 12 weeks.

10. The method of claim I, wherein said cartilage construct is inserted subcutaneous ly into the animal host in step b).

1 1. The method of claim I, wherein the cartilage construct is maintained within the animal host for at least about 12 weeks.

12. A cartilage construct synthesized by the method of claim 1.

13. A method of replacing damaged or deficient cartilage in a subject, said method comprising inserting into said subject the cartilage construct of claim 12.

14. The method of claim 13, wherein said the chondrocytes cultured in vitro are obtained from said subject.

15. The method of claim 13, wherein said damaged or deficient cartilage is selected from the group consisting of cartilage of the ear, cartilage of the nose, cartilage of the joints, cartilage of the knee, cartilage of the hip, cartilage of the shoulder, cartilage of the rib cage, cartilage of the larynx, cartilage of the trachea, and cartilage between vertebral discs.