**7.4.2 Histology**

148 Modern Arthroscopy

Fig. 16. 34-year-old female from Fig 22. Second-look arthroscopy from the lateral patella facet and lateral trochlear showed tufts of cartilage forming at each individual drill hole. Upon histological stains, notice the red staining with Safranin-O representing proteoglycans

and the brown staining of collagen type II diffuse throughout the regenerated tissue.

Collagen type I stain is minimal and localized near the superficial layers.

Histologic samples were stained as follows: hematoxylin-eosin (H&E) stain was used to visualize overall morphology, Safranin-O was used to highlight proteoglycans, immunohistochemistry staining with anti–collagen type I mouse Ab I-8H5 stain (catalog No. CP 17; Calbiochem Merck, Darmstadt, Germany) was used to highlight collagen type I, and immunohistochemistry staining with anti–collagen type II mouse monoclonal antibody Ab 3 (clone 6B3) (catalog No. MAB8887; Millipore, Billerica, MA) was used to highlight collagen type II. Optimal dilution and predigestion with pepsin were determined by the investigator with the protocol being saved by use of software of an automated immunohistochemical slide preparation system (Ventana Benchmark; Ventana Medical Systems, Tucson, AZ).

Cases 1 and 2 with gross grade IV kissing lesions are presented with multiple biopsy specimens and histologic analyses after second-look arthroscopy. Cases 3, 4, and 5 are patients with smaller isolated lesions.

**Case 1**: Biopsy was performed 22 months after the initial surgery in a 49-year-old woman with a varus deformity who underwent debridement, subchondral drilling, and an open wedge high tibial osteotomy with Tomofix fixation (Fig 17).

Approximately 80% of the weight-bearing medial compartment had grade III and IV lesions. Weight-bearing radiographs at 8 and 18 months showed reappearance of the medial femorotibial joint space. Second-look arthroscopy of the regenerated cartilage showed a stable, smooth surface with no delamination. On probing, the regenerated cartilage had the same consistency as the surrounding normal cartilage. The second-look images and biopsy specimens are included in Fig 18.

Immunohistochemistry staining was performed to assess the collagen type I and type II content of the biopsy specimens. Specimens from the medial femoral condyle and medial tibial plateau showed the presence of collagen type I confined to the superficial layer. Collagen type II was present throughout the deeper layers. These are features of hyaline cartilage as opposed to fibrocartilage (Fig 18). Fig 19 shows a higher magnification of the histological sections from the medial tibial plateau.

zone.

Articular Cartilage Regeneration with Stem Cells 151

One full-thickness biopsy specimen from the medial tibial plateau captured a drill hole and adjacent bone (Fig 20). This sample shows full-thickness regenerated articular cartilage with a fairly smooth articular surface, subchondral bone, and marrow space. A streaming, linear pattern of chondrocytes is seen arising from the subchondral bone region from the area of previous subchondral drilling. Incidental findings of cartilage clusters with early ossification are also seen within the tract. The chondrocytes are involved in ongoing remodelling and are present beneath the calcified cartilage layer. A biopsy specimen from the medial femoral condyle of the patient also showed the presence of a previous drill hole lined with chondrocytes. This sample illustrated an area of regeneration undergoing ossification, showing re-establishment of the calcified cartilage layer and the tidemark (Fig 21). The tidemark is seen as an undulating basophilic line on routine H&E staining and is the point at which the articular cartilage becomes calcified. The subchondral drill defect area is replaced by the presence of chondrocytes with varying degrees of maturation surrounded by a ground substance matrix. In addition, ossification is evident with new trabecular bone formation. Ossification changes were not seen over the new hyaline cartilage formation

Fig. 19. Biopsy from the medial tibial plateau in case 1 showing the presence of

specific stains (Original magnification X 100).

chondrocytes and collagen fibres aligning to the axis of weight transmission with their

Fig. 18. Medial tibial plateau (MTP), medial femoral condyle (MFC) and intercondylar notch (ICN) biopsy results at 22 months after surgery. Biopsies from the MTP and MFC with H&E staining illustrate columnar morphology of cells with pale background. Safranin-O staining highlights abundance of proteoglycans throughout the regenerated cartilage layer. Collagen type I staining was limited to the superficial layer except in the non-weight-bearing ICN biopsy which showed a higher percentage of collagen type I and a disorganized pattern of healing. Collagen type II was concentrated in the deeper layers.

Fig. 18. Medial tibial plateau (MTP), medial femoral condyle (MFC) and intercondylar notch (ICN) biopsy results at 22 months after surgery. Biopsies from the MTP and MFC with H&E staining illustrate columnar morphology of cells with pale background. Safranin-O staining highlights abundance of proteoglycans throughout the regenerated cartilage layer. Collagen type I staining was limited to the superficial layer except in the non-weight-bearing ICN biopsy which showed a higher percentage of collagen type I and a disorganized pattern of

healing. Collagen type II was concentrated in the deeper layers.

One full-thickness biopsy specimen from the medial tibial plateau captured a drill hole and adjacent bone (Fig 20). This sample shows full-thickness regenerated articular cartilage with a fairly smooth articular surface, subchondral bone, and marrow space. A streaming, linear pattern of chondrocytes is seen arising from the subchondral bone region from the area of previous subchondral drilling. Incidental findings of cartilage clusters with early ossification are also seen within the tract. The chondrocytes are involved in ongoing remodelling and are present beneath the calcified cartilage layer. A biopsy specimen from the medial femoral condyle of the patient also showed the presence of a previous drill hole lined with chondrocytes. This sample illustrated an area of regeneration undergoing ossification, showing re-establishment of the calcified cartilage layer and the tidemark (Fig 21). The tidemark is seen as an undulating basophilic line on routine H&E staining and is the point at which the articular cartilage becomes calcified. The subchondral drill defect area is replaced by the presence of chondrocytes with varying degrees of maturation surrounded by a ground substance matrix. In addition, ossification is evident with new trabecular bone formation. Ossification changes were not seen over the new hyaline cartilage formation zone.

Fig. 19. Biopsy from the medial tibial plateau in case 1 showing the presence of chondrocytes and collagen fibres aligning to the axis of weight transmission with their specific stains (Original magnification X 100).

Articular Cartilage Regeneration with Stem Cells 153

The intercondylar notch sample was an area of previous roof-plasty and notchplasty with abrasion chondroplasty. This represents an area that is non–weight-bearing. Histologic biopsy examination here showed a mixed tissue with fibrocartilage and hyaline-like cartilage: the predominance of collagen type I, less collagen type II, and chondrocytes arranged in a more disorganized pattern. We believe that the failure of the chondrocytes to exhibit a linear streaming pattern, as seen from the medial tibial plateau and medial femoral condyle biopsy specimens, is due to the absence of stimulation from weight-bearing forces

**Case 2:** A chondral core biopsy specimen was taken 26 months after surgery in a 34-year-old woman with previous multiple open surgeries for recurrent dislocation of the patella as an adolescent. She underwent arthroscopic debridement, lateral patella release, and subchondral drilling. There were grade III and IV lesions over the entire patellofemoral joint. Preoperative merchant-view radiographs showed severe patellofemoral osteoarthritis, a large medial trochlear osteophyte, and absence of the lateral patellofemoral joint space (Fig 22). During arthroscopic surgery, the medial trochlear osteophyte was burred and subchondral drilling was performed on the entire patellofemoral joint. An immediate postoperative radiograph showed evidence of subchondral drilling. Radiographs at 6 months and 2 years showed progressive reappearance of the lateral patellofemoral joint space (Fig 22). Fig 16 contains results of the second-look arthroscopy with chondral core biopsy specimens. The lateral patella facet and lateral trochlear areas showed tufts of cartilage between areas devoid of cartilage. In contrast, the medial trochlear area that underwent the removal and burring of a large osteophyte followed by subchondral drilling

showed complete coverage by newly formed articular cartilage.

Fig. 22. 34-year-old female with recurrent dislocation of her patella as an adolescent. Immediate postoperative radiographs showed evidence of subchondral drilling while radiographs at 6 months and 2 years showed a progressive reappearance of the lateral

on the intercondylar notch (Fig 18).

patellofemoral articulation.

Fig. 20. **(**A) H&E sample as in Figure 18 from the medial tibial plateau managed to biopsy an area of previous drilling. (B) Upon higher magnification, chondrocytes appeared deep to the subchondral bone with areas of ossification. Notice the chondrocytes below the subchondral bone region beginning as immature cells (short arrow) and progressing toward the joint surface as mature chondrocytes in rows (long arrow). (C) Upon staining with Safranin-O, there was a high concentration of proteoglycans at the base of the regenerated tissue represented as red coloration (double arrow).

Fig. 21. A biopsy from the medial femoral condyle illustrating ossification of regenerated cartilage and the re-establishment of the calcified cartilage (white arrow) layer with tidemark (black arrow). Note the presence of chondrocytes below the calcified cartilage (double arrow) with areas of ossification.

Fig. 20. **(**A) H&E sample as in Figure 18 from the medial tibial plateau managed to biopsy an area of previous drilling. (B) Upon higher magnification, chondrocytes appeared deep to

subchondral bone region beginning as immature cells (short arrow) and progressing toward the joint surface as mature chondrocytes in rows (long arrow). (C) Upon staining with Safranin-O, there was a high concentration of proteoglycans at the base of the regenerated

Fig. 21. A biopsy from the medial femoral condyle illustrating ossification of regenerated cartilage and the re-establishment of the calcified cartilage (white arrow) layer with tidemark (black arrow). Note the presence of chondrocytes below the calcified cartilage

the subchondral bone with areas of ossification. Notice the chondrocytes below the

tissue represented as red coloration (double arrow).

(double arrow) with areas of ossification.

The intercondylar notch sample was an area of previous roof-plasty and notchplasty with abrasion chondroplasty. This represents an area that is non–weight-bearing. Histologic biopsy examination here showed a mixed tissue with fibrocartilage and hyaline-like cartilage: the predominance of collagen type I, less collagen type II, and chondrocytes arranged in a more disorganized pattern. We believe that the failure of the chondrocytes to exhibit a linear streaming pattern, as seen from the medial tibial plateau and medial femoral condyle biopsy specimens, is due to the absence of stimulation from weight-bearing forces on the intercondylar notch (Fig 18).

**Case 2:** A chondral core biopsy specimen was taken 26 months after surgery in a 34-year-old woman with previous multiple open surgeries for recurrent dislocation of the patella as an adolescent. She underwent arthroscopic debridement, lateral patella release, and subchondral drilling. There were grade III and IV lesions over the entire patellofemoral joint. Preoperative merchant-view radiographs showed severe patellofemoral osteoarthritis, a large medial trochlear osteophyte, and absence of the lateral patellofemoral joint space (Fig 22). During arthroscopic surgery, the medial trochlear osteophyte was burred and subchondral drilling was performed on the entire patellofemoral joint. An immediate postoperative radiograph showed evidence of subchondral drilling. Radiographs at 6 months and 2 years showed progressive reappearance of the lateral patellofemoral joint space (Fig 22). Fig 16 contains results of the second-look arthroscopy with chondral core biopsy specimens. The lateral patella facet and lateral trochlear areas showed tufts of cartilage between areas devoid of cartilage. In contrast, the medial trochlear area that underwent the removal and burring of a large osteophyte followed by subchondral drilling showed complete coverage by newly formed articular cartilage.

Fig. 22. 34-year-old female with recurrent dislocation of her patella as an adolescent. Immediate postoperative radiographs showed evidence of subchondral drilling while radiographs at 6 months and 2 years showed a progressive reappearance of the lateral patellofemoral articulation.

Articular Cartilage Regeneration with Stem Cells 155

Findings from second-look arthroscopy presented in the above 5 case series confirmed that it was possible to replicate the results of our animal model (Saw et al, 2009) in the human knee joint using a combination of postoperative intraarticular injections of autologous PBPC with HA after arthroscopic subchondral drilling. This method uses marrow stimulation to create an autologous scaffold that is subsequently seeded with both intraarticular injections of autologous PBPC and in-situ progenitor cells from the underlying marrow (Fig 24).

In a typical chondral lesion of the medial femoral condyle (Fig 24) measuring 2cm x 2cm,

Fig. 24. Chondrogenesis: Contained lesion. (A) A blood clot scaffold is formed after

(D) The final result is the formation of a new layer of articular cartilage with good integration to the surrounding tissues. The ossification of the chondrocytes below the articular cartilage results in repair of the subchondral bone with re-establishment of the

subchondral drilling and abrasion chondroplasty between the drill holes. The surrounding articular cartilage is normal with the underlying calcified cartilage layer and subchondral bone. (B) Injection of fresh PBPC plus HA 1 week after surgery results in the homing of the PBPC into the blood clot scaffold. (C) The PBPC residing in the osteochondral junction and blood clot scaffold gradually transform to chondrocytes. HA helps to reduce inflammation and provide raw material for chondrogenesis. The injected PBPC exert paracrine effects and recruit in-situ progenitor cells to assist in chondrogenesis. Repeated injections of PBPC plus HA enable more cells to be recruited into the chondral defect and enhance chondrogenesis.

**7.4.3 Chondrogenesis with PBPC and HA** 

chondrogenesis progresses as described in Fig 24.

**7.4.4 Contained lesion** 

calcified cartilage layer.

**Case 3:** A biopsy specimen was obtained 1 year after surgery from a 52-year-old woman with an isolated grade IV lesion of the lateral femoral condyle measuring 2 X 1 cm.

**Case 4:** A biopsy specimen was obtained 10 months after surgery in a 43-year-old woman with lateral patellar maltracking. There was a grade III/IV defect measuring 2.5 X 3.5 cm over the lateral patella facet. Lateral patella release was performed in addition to subchondral drilling.

**Case 5:** Biopsy was performed at 18 months after surgery in a 19-year-old man with lateral patellar maltracking and a lateral trochlear grade IV lesion measuring 0.8 cm in diameter. Subchondral drilling was performed followed by lateral patellar release (Fig 23).

Chondral core biopsy specimens in cases 2, 3, 4, and 5 with histologic staining with H&E showed columnar morphology of cells with a pale blue ground substance. Safranin-O showed intense orange / red staining of the newly regenerated cartilage zone throughout the regenerated cartilage layer with a propensity toward the deeper areas of cartilage above the subchondral bone. The matrix also showed a predominance of collagen type II deposits, whereas collagen type I was minimal and located mostly over the superficial regions of the articular surface. These compositional results are features of hyaline as opposed to fibrocartilage (Kang et al, 2008; Saw et al, 2009 & Lee et al, 2007).

Fig. 23. Second-look arthroscopy and histological images of a lesion from the lateral femoral condyle (Case 3), a lesion from the lateral patella facet (Case 4) and a lesion from the lateral trochlear (Case 5).
