DeNovo^® NT Natural Tissue Graft

Articular cartilage can be damaged by injury or normal wear and tear of the joint. Due to its inherent lack of blood supply, this tissue is not able to heal well, and surgical efforts to repair damaged cartilage continue to evolve. Articular cartilage undergoes substantial age-related changes with respect to its chemical composition, tissue structure and mechanical properties, which manifest themselves with markedly different tissue repair and restoration capabilities. Therefore, to develop effective surgical treatments, it is critical to understand the effects that age has on the biological properties and tissue restoration capabilities of cartilage and determine the mechanism underlying these changes.

Articular cartilage was harvested from the knee joints of juvenile (four months) and adult (six-eight years) bovines and prepared into ring-shaped discs. These discs were cultured for four weeks to monitor migration of cartilage cells (chondrocytes), preservation of components that help the cartilage maintain its elasticity (glycosaminoglycans or (GAGs) and formation of new cartilage tissue. The number of cartilage cells in a given amount of tissue (cell density) and proliferative activity of these cells were also compared between the two age groups. Additionally, age-related changes in the gene expression of the chondrocytes were analyzed using Affymetrix GeneChip array.

Compared with adult cartilage, juvenile bovine cartilage demonstrated a significantly:

• Greater cell density (Fig. 1)
• Higher cell proliferation rate (Fig. 2)
• Increased number of migrated and proliferated cells showing typical chondrocyte feature (Figs. 3 and 4)
• Elevated GAG content in both native and cultured tissues (Fig. 5).

During four weeks in culture, juvenile cartilage generated new cartilage tissues (Fig. 6) exhibiting pronounced labeling for proteoglycan and type II collagen(the collagen found in articular cartilage) but not type I collagen (the collagen found in scar tissue)(Fig. 7). Adult cartilage exhibited no obvious changes.

Over 19,000 genes were analyzed to identify distinctive gene expression profiles between juvenile and adult cartilage tissues. Genes primarily responsible for cartilage growth and expansion (such as COL2A1, COL9A1, TGFB3, MMP2 and MMP14) were increased (up-regulated) in juvenile cartilage, whereas the genes mostly involved in structural stability (such as COMP, FN1, BMP2, TIMP2 and TIMP3) were up-regulated in adult cartilage.

Fig. 1. Juvenile bovine cartilage had a significantly higher cell density than adult cartilage (mean with 95% confidence interval (CI), P = 0.004).

Fig. 2. Juvenile bovine chondrocytes proliferated significantly faster than adult chondrocytes by day3 (mean with 95% CI, P = 0.001). Day 1 results indicate a similar assay baseline for both age groups.

A.		B.
C.		D.

Fig. 3. Cell migration from juvenile (A, 20x; C, 40x) and adult (B, 20x; D, 40x) bovine cartilage. Migrating cells from both tissues exhibited typical chondrocyte features.

Fig. 4. A significantly greater number of migrated and proliferated cells were detected in the juvenile cartilage culture than in the adult cartilage culture (mean with 95% CI, P = 0.006).

Fig. 5. Both native and cultured juvenile bovine cartilage contained a significantly greater amount of GAG than the adult cartilage (mean with 95% CI, p<0.05).

	Adult Cartilage		Juvenile Cartilage
DAY 0
DAY 28

Fig. 6. Only juvenile cartilage was able to generate new cartilaginous tissues after 4 weeks of culture (2.5x).

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C.		D.

Fig. 7. The new tissues generated from juvenile bovine cartilage exhibited strong labeling of type II collagen and proteoglycan with no detectable type I collagen. (A) H & E, (B) Safranin-O, (C) Type II collagen and (D) Type I collagen.

As the first comprehensive comparison between juvenile and adult bovine articular cartilage at the tissue, cellular and molecular level's, our results strongly indicate that juvenile cartilage possesses greater regenerative capability and superior chondrogenic activity than adult cartilage. Additionally, the chondrocyte age-related differential gene expression supports enhanced tissue restoration potential of juvenile cartilage.

1. Hui Liu, Zhixing Zhao, Rhonda B. Clarke, Jizong Gao, Ian R. Garrett and Ed E.C. Margerrison. Enhanced tissue regeneration potential of juvenile articular cartilage, The American Journal of Sports Medicine 41(11):2658-2667, 2013. *Animal study results are not necessarily predictive of human clinical results.

Juvenile human articular cartilage taken from the knee joint of an 11-year-old donor was cultured in vitro to monitor chondrocyte migration and new cartilage tissue formation. Similar results were obtained as from the juvenile bovine cartilage culture:

• Cells started migrating out of the minced juvenile human articular cartilage by day 5 of culture. The migrated cells demonstrated typical chondrocyte features (Fig. 1).
• The migrated cells accumulated and proliferated along the edges of the original cartilage pieces (Fig. 2).
• The migrated cells generated new tissue (extracellular matrix or ECM) along the edges of the original cartilage tissue, which fused the adjacent cartilage pieces together (Fig. 3).
• The newly formed tissue between the original cartilage pieces became denser and stronger over time, with multiple layers of rounded cells embedded in the newly generated ECM (Fig. 4).

A.		B.
C.		D.

Fig. 1. Cells started migrating out of juvenile human articular cartilage at day 5 of culture (A: 20x, B: 40x). The migrated cells show the typical chondrocyte features (C: 10x, D: 40x).

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B.

Fig. 2. The migrated chondrocytes accumulated and proliferated along the edges of the original cartilage pieces (A:10x, B: 5x).

A.		B.
C.		D.

Fig. 3. The new ECM structures generated by the migrated chondrocytes formed inter-piece connections, fusing the adjacent cartilage pieces together (A: 2.5x, B: 5x, C:10x, D: 20x).

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B.

C.

Fig. 4. The newly formed cartilaginous tissue became denser and stronger over time as shown in- A(day 15 of culture,5x), B(day 30 of culture,5x) and C(a higher magnification view of the new tissue structure in B, showing rounded cells embedded in the newly generated ECM(40x).)

Adams SB, Yao JQ, Schon LC. Particulated Juvenile Articular Cartilage Allograft Transplantation for Osteochondral Lesions of the Talus. Tech Foot Ankle Surg Jun 2011; 10(2):92-98.
A treatment algorithm and detailed description of surgical technique for treatment of talar lesions with DeNovo NT Graft. This paper describes 12-month postoperative images of a 24-year-old male with near complete resolution of talar edema and formation of cartilage surface.
Link to Abstract: http://journals.lww.com/techfootankle/Abstract/2011/06000/Particulated_Juvenile_Articular_Cartilage.10.aspx

Bleazy S and Brigido S. Reconstruction of Complex Osteochondral Lesions of the Talus with Cylindrical Sponge Allograft and Particulate Juvenile Cartilage Graft: Provisional Results with a Short-Term Follow up. Foot Ankle Spec Oct 2012; 5:300-305 (first published online Aug 30, 2012).
Retrospective review of 7 patients with full-thickness cartilage defects treated with medial malleolar osteotomy, subchondral allograft and DeNovo NT Graft. All 7 patients showed clinically significant improvement in pain and activity scales 6 months postoperatively.
Link to Abstract: http://fas.sagepub.com/content/5/5/300

Bonner KF, Daner W, Yao JQ. 2-Year Postoperative Evaluation of a Patient with a Symptomatic Full-Thickness Patellar Cartilage Defect Repaired with Particulated Juvenile Cartilage Tissue. J Knee Surg Jun 2010; 23(2):109-14.
Case report of a 36-year-old male with a full-thickness chondral defect of the patella treated with DeNovo NT Graft. At 2 years postoperative, MRI demonstrates full defect fill and resolution of subchondral bone edema, and KOOS and IKDC outcomes scores are substantially improved.
Link to Abstract: http://www.ncbi.nlm.nih.gov/pubmed/21141688

Coetzee JC, Giza E, Schon LC, Berlet GC, Neufeld S, Stone RM, Wilson EL. Treatment of Osteochondral Lesions of the Talus With Particulated Juvenile Cartilage. Foot & Ankle Intl, Apr 2013; 34(9):1205-1211
Case series of 24 ankle surgeries, implanted with DeNovo NT Graft, for symptomatic chondral lesions. At an average follow up time of 16 months, 78% had good to excellent scores on the AOFAS scale with one partial revision.
Link to Abstract: http://fai.sagepub.com/content/early/2013/04/10/1071100713485739.abstract

Cole, B, Farr J, Tabet S, Gold G, Carlson C, Margerrison E. Clinical, Radiographic and Histological Outcomes following Cartilage Repair with Particulated Juvenile Articular Cartilage: A 2-Year Prospective Study. Am J Sports Med, in press
A prospective study of 25 subjects treated with DeNovo NT for cartilage lesions. Follow up at 2 years post surgery demonstrated significant improvement in clinical outcome scores, MRI T2 values approximating normal cartilage, and histology results showing repair tissue inclusive of hyaline cartilage and excellent integration.

Farr J, Cole B, and Sherman S, Karas V. Particulated Articular Cartilage: CAIS and DeNovo NT. J Knee Surg Mar 2012; 25(1):23-29.
Review of surgical technique and preliminary data for DeNovo NT Graft and Cartilage Autograft Implantation System (CAIS). This paper summarizes previously published findings of clinical improvement and defect resolution via MRI.
Link to Abstract: http://www.ncbi.nlm.nih.gov/pubmed?term=Farr%20CAIS%20DeNovo%20NT

Farr J and Yao JQ. Chondral Defect Repair with Particulated Juvenile Cartilage Allograft. Cartilage Oct 2011; 2(4):346-353 (first published online Jul 15, 2011).
Case study of 4 patients with chondral lesions of the femoral condyle or trochlear treated with DeNovo NT Graft. 2-year follow up demonstrated clinically significant improvements in KOOS, IKDC subjective and VAS pain scores. MRI showed good defect fill that persists at least two years.
Link to Abstract: http://car.sagepub.com/content/2/4/346.abstract?rss=1

Hatic SO and Berlet GC. Particulated Juvenile Articular Cartilage Graft for Treatment of Osteochondral Lesions of the Talus. Foot Ankle Spec Dec 2010; 3(6):361-364.
A review of treatment options for osteochondral lesions of the talus and a description of the surgical technique for implanting DeNovo NT Graft.
Link to Abstract: http://fas.sagepub.com/content/3/6/361

Kruse DL, Ng A, Paden M, Stone PA. Arthroscopic DeNovo NT Juvenile Allograft Cartilage Implantation in the Talus: A Case Presentation. J Foot Ankle Surg Mar-Apr 2012; 51(2): 218-21.
A case study of a 30-year-old female with a grade 4 talar osteochondral lesion treated with DeNovo NT Graft. The patient was pain free by 6 months postoperation and returned to full activity. The patient was interviewed again at 24 months postoperation with the same results.
Link to Abstract: http://www.ncbi.nlm.nih.gov/pubmed/22138532

Liu H, Zhao Z, Clarke RB, Gao J, Garrett IR, Margerrison EEC. Enhanced Tissue Regeneration Potential of Juvenile Articular Cartilage. AJSM Nov 2013; 41(11):2658-2667.
A comparison of the biological properties and tissue regeneration capabilities of juvenile and adult bovine articular cartilage. Results suggest an enhanced regenerative potential of juvenile cartilage tissue in the restoration of damaged articular cartilage.
Link to Abstract: http://ajs.sagepub.com/content/early/2013/09/16/0363546513502945

Tompkins M, Hamann JC, Diduch DR, Bonner KF, Hart JM, Gwathmey W, Milewski MD, Gaskin CM. Preliminary Results of a Novel Single Stage Cartilage Restoration Technique: Particulated Juvenile Articular Cartilage Allograft for Chondral Defects of the Patella. Arthroscopy Oct 2013; 29(10):1661-1670.
Case series of 15 knee surgeries for grade 4 lesions of the patella treated with DeNovo NT. At an average follow up time of 28 months, the mean fill was 89% and the ICRS cartilage repair assessment score on MRI was “nearly normal” for all subjects. Clinical outcomes scores for IKDC and KOOS scales were comparable to those published for ACI.
Link to Abstract: http://www.ncbi.nlm.nih.gov/pubmed/23876608