**7.4.9 Role of weight-bearing**

Our biopsy specimens from cases with subchondral drilling followed by postoperative intraarticular injections of PBPC in combination with HA showed histologic features of hyaline cartilage with anti–collagen type I stain (used to highlight collagen type I), anti– collagen type II stain (used to highlight collagen type II), and Safranin-O stain (used to highlight proteoglycans), with the exception of one histologic sample showing mixed cartilage. This biopsy specimen was from an area of abrasion notchplasty, which represented a non–weight-bearing region. Comparison of biopsy specimens from this non– weight-bearing area to those from a weight-bearing area in the same patient has led us to theorize that early partial-weight-bearing is essential for the regeneration and alignment of collagen type II (Fig 18).

#### **7.4.10 Articular cartilage imaging of the knee**

Articular cartilage is visible on most standard MRI sequences as a band of intermediate to high signal covering the articular aspect of the bone. Non-injured articular cartilage normally shows a continuous subchondral dark line (low-signal) below the articular cartilage. This dark line likely represents the layer of calcified cartilage and the associated subchondral bone plate. It may be accentuated by chemical shift artifact (of water and fat molecules in the same voxel canceling their respective signals thereby resulting in signal loss). Fig 41 illustrates the articular cartilage and subchondral bone on a sagittal PD image. Evaluating postoperative MRIs, we assessed the restoration of the dark line as evidence of calcified cartilage with subchondral bone healing and fill of the defect as indicative of cartilage regeneration.

Our current imaging preference for assessing chondral lesions of the knee joint utilize 2D PD and PDFS sequences using a high field (1.5T) extremity MRI (GE Medical Systems) – Fig 42. This has the benefits of improved signal to noise and higher resolution. Fig 43 showing examples of chondral lesions seen following MRI scan with arthroscopic correlation (Fig 44). Our earlier cartilage images were obtained by an open MRI system operating at 0.35T (Magnetom C!, Siemens Medical Solutions, Erlangen, Germany) using an extremity receive coil.

MRI scans were utilized preoperatively as part of the diagnostic work up and postoperatively to monitor healing of the chondral defects. We performed MRI scans at

**7.4.8 The importance of PBPC + HA as adjuvant therapy following ideal subchondral**

Case 6 emphasizes on the importance of the improved technique of ideal drilling in an uncontained lesion. Cases 7 & 8 illustrates the importance of postoperative intraarticular

Possible reasons as to why the microfracture technique has not been successful in achieving consistent coverage with hyaline cartilage can be explained by what is seen on second-look arthroscopy in cases 6, 7 and 8. Firstly, microfractures are usually placed 3 to 5 mm apart and do not penetrate much deeper than the calcified cartilage layer. Microfractures placed more superficially and further apart as compared to ideal subchondral drilling explains one of the possible reasons why the microfracture technique is inconsistent in producing satisfactory articular cartilage repair. Secondly, like the animal model in Fig 9, without postoperative adjunct therapy with PBPC + HA, the regenerated tissue will always be of inferior quality.

Our biopsy specimens from cases with subchondral drilling followed by postoperative intraarticular injections of PBPC in combination with HA showed histologic features of hyaline cartilage with anti–collagen type I stain (used to highlight collagen type I), anti– collagen type II stain (used to highlight collagen type II), and Safranin-O stain (used to highlight proteoglycans), with the exception of one histologic sample showing mixed cartilage. This biopsy specimen was from an area of abrasion notchplasty, which represented a non–weight-bearing region. Comparison of biopsy specimens from this non– weight-bearing area to those from a weight-bearing area in the same patient has led us to theorize that early partial-weight-bearing is essential for the regeneration and alignment of

Articular cartilage is visible on most standard MRI sequences as a band of intermediate to high signal covering the articular aspect of the bone. Non-injured articular cartilage normally shows a continuous subchondral dark line (low-signal) below the articular cartilage. This dark line likely represents the layer of calcified cartilage and the associated subchondral bone plate. It may be accentuated by chemical shift artifact (of water and fat molecules in the same voxel canceling their respective signals thereby resulting in signal loss). Fig 41 illustrates the articular cartilage and subchondral bone on a sagittal PD image. Evaluating postoperative MRIs, we assessed the restoration of the dark line as evidence of calcified cartilage with subchondral bone healing and fill of the defect as indicative of

Our current imaging preference for assessing chondral lesions of the knee joint utilize 2D PD and PDFS sequences using a high field (1.5T) extremity MRI (GE Medical Systems) – Fig 42. This has the benefits of improved signal to noise and higher resolution. Fig 43 showing examples of chondral lesions seen following MRI scan with arthroscopic correlation (Fig 44). Our earlier cartilage images were obtained by an open MRI system operating at 0.35T (Magnetom C!, Siemens Medical Solutions, Erlangen, Germany) using an extremity receive

MRI scans were utilized preoperatively as part of the diagnostic work up and postoperatively to monitor healing of the chondral defects. We performed MRI scans at

**drilling**

injections of PBPC + HA.

**7.4.9 Role of weight-bearing**

collagen type II (Fig 18).

cartilage regeneration.

coil.

**7.4.10 Articular cartilage imaging of the knee**

shorter intervals for our first 10 patients. Scans were performed on the first postoperative day as a baseline to document the chondral defect after debridement and subchondral drilling. Serial studies were then collected in the postoperative period (at 6, 12, 18, 24 months and beyond) to evaluate the filling of the defect by regenerated cartilage and changes in the subchondral bone.

Fig. 41. Articular cartilage as depicted by sagittal PD image at the tibial plateau. Black arrow showing the surface of the tibial plateau articular cartilage and red arrow showing the "Black line" that separates the articular cartilage from the subchondral bone. This layer correlates with the tidemark / calcified cartilage / subchondral bone plate layers.

Fig. 42. A patient with her right knee in a high field extremity (1.5T) MRI (GE Medical Systems).

Articular Cartilage Regeneration with Stem Cells 169

MRI scans performed on the first postoperative day showed the chondral defects as well delineated from surrounding healthy cartilage. The chondral defects were bare or partially filled in with blood clot. Drill tracts and subchondral marrow edema were clearly observed (Figures 45 to 47). Subchondral bone is disrupted, i.e. there is loss of the continuity of the subchondral black line. Over the course of two years, filling-in of the chondral defects by material of similar or same signal as articular cartilage was observed. Re-establishment of subchondral black line paralleled the progressive resolution of

Serial MRI scans of our first patient undergoing this treatment are presented in Figure 45. After full debridement and drilling, the chondral defect is well visualized as a bare area partially filled by clot (Figure 45A). The low signal subchondral bone is disrupted and low signal drill tracts are visualized within the subchondral marrow edema. The disruption of the subchondral dark line represents a conduit through the calcified cartilage layer. Serial scans showed progressive filling of chondral defects by material of similar appearance as articular cartilage, resolution of marrow edema and reappearance of the continuous subchondral dark line (Figure 45B). At one to two years, the calcified cartilage, as depicted by the low signal band, becomes almost as thick at the site of drilling as in the surrounding

healthy areas. (Figure 45D). Figures 46 and 47 present an additional patient.

Fig. 45. Serial MRI evaluation (STIR images at 0.35T). Postoperative (Post-op): note the disruption of the subchondral dark line (red arrow). (B) 2 months: partial resolution of marrow edema and reappearance of the continuous subchondral dark line (red arrow). (C) 10 months: almost complete resolution of marrow edema and filling of defect. (D) 20 months: complete healing of defect and re-establishment of subchondral dark line

representing healing of the calcified cartilage layer.

marrow edema.

Fig. 43. Images of chondral lesions demonstrated by extremity high field MRI at 1.5T. (A) Chondral flap tear of the medial femoral condyle (white arrow) with red arrow showing a delaminated chondral lesion over the lateral femoral condyle (PDFS). (B) The corresponding chondral lesion of the lateral femoral condyle (red arrow) on a sagittal PD image.

Fig. 44. Corresponding arthroscopic view of the lesions from the medial femoral condyle (MFC) and lateral femoral condyle (LFC) as in Fig 43.

Fig. 43. Images of chondral lesions demonstrated by extremity high field MRI at 1.5T. (A) Chondral flap tear of the medial femoral condyle (white arrow) with red arrow showing a delaminated chondral lesion over the lateral femoral condyle (PDFS). (B) The corresponding

Fig. 44. Corresponding arthroscopic view of the lesions from the medial femoral condyle

(MFC) and lateral femoral condyle (LFC) as in Fig 43.

chondral lesion of the lateral femoral condyle (red arrow) on a sagittal PD image.

MRI scans performed on the first postoperative day showed the chondral defects as well delineated from surrounding healthy cartilage. The chondral defects were bare or partially filled in with blood clot. Drill tracts and subchondral marrow edema were clearly observed (Figures 45 to 47). Subchondral bone is disrupted, i.e. there is loss of the continuity of the subchondral black line. Over the course of two years, filling-in of the chondral defects by material of similar or same signal as articular cartilage was observed. Re-establishment of subchondral black line paralleled the progressive resolution of marrow edema.

Serial MRI scans of our first patient undergoing this treatment are presented in Figure 45. After full debridement and drilling, the chondral defect is well visualized as a bare area partially filled by clot (Figure 45A). The low signal subchondral bone is disrupted and low signal drill tracts are visualized within the subchondral marrow edema. The disruption of the subchondral dark line represents a conduit through the calcified cartilage layer. Serial scans showed progressive filling of chondral defects by material of similar appearance as articular cartilage, resolution of marrow edema and reappearance of the continuous subchondral dark line (Figure 45B). At one to two years, the calcified cartilage, as depicted by the low signal band, becomes almost as thick at the site of drilling as in the surrounding healthy areas. (Figure 45D). Figures 46 and 47 present an additional patient.

Fig. 45. Serial MRI evaluation (STIR images at 0.35T). Postoperative (Post-op): note the disruption of the subchondral dark line (red arrow). (B) 2 months: partial resolution of marrow edema and reappearance of the continuous subchondral dark line (red arrow). (C) 10 months: almost complete resolution of marrow edema and filling of defect. (D) 20 months: complete healing of defect and re-establishment of subchondral dark line representing healing of the calcified cartilage layer.

Articular Cartilage Regeneration with Stem Cells 171

Fig. 47. Sagittal PD MRI (0.35T) of patient in Figure 46. (A) Postoperative (Post-op) MRI: chondral lesions at the lateral patella and lateral femoral chondyle (arrows). Note the disruption to the continous low signal calcified cartilage layer at both sites. (B) MRI at one year after surgery showing the re-establishment of the subchondral calcified cartilage layer

The re-establishment of the calcified cartilage layer and healing of the subchondral bone are important MRI features of articular cartilage regeneration in our series. This is accompanied by filling of the chondral defect. Following arthroscopic subchondral drilling, MRI images revealed extensive marrow edema and interruption of the calcified cartilage layer together with the underlying subchondral bone. This is shown as disruption of the low signal subchondral dark line. As the injected PBPC seed the blood clot scaffold in the presence of HA, chondrogenesis is initiated with the formation of chondrocytes which then occupy the drill holes. This process is gradually replaced by bone resulting in subchondral bone repair. Gradual resolution of marrow edema is observed. The subchondral dark line progressively re-appears on MRI scans indicating the re-establishment of the calcified cartilage layer and healing of the subchondral bone (Figures 45 to 47). This evidence is provided from the histology shown on Figures 20 and 21. Depending on whether the lesion is a contained or uncontained lesion, chondrogenesis follows the gradual re-appearance of this subchondral

Since starting clinical trials in 2007, 223 cartilage regeneration cases have been performed on 205 patients. Cases were varied including 38 cases involving isolated cartilage lesions, 92 cases involving multiple cartilage lesions, 54 cases involving patellofemoral cartilage lesions, 7 cases involving concomitant lower limb realignment procedures, and 32 cases involving ligament reconstruction. When evaluating two-year clinical outcomes, 24 months has passed for 155 of these 223 cases. Within this group, 52 cases have preoperative, 12-month, and 24-month IKDC values available for clinical outcome evaluation. This group had a preoperative IKDC average of 50.5, a 12-month IKDC

(long arrows) together with articular cartilage regeneration (short arrows).

dark line on serial MRI scans (Figures 24 and 26).

**7.4.11 Phase I study clinical outcomes**

Fig. 46. A 40-year-old woman with patellar dislocation (STIR images at 0.35T). (A) Preoperative MRI with evidence of a delaminating articular cartilage injury and medial patellar femoral ligament (MPFL) injury. The injury was treated with arthroscopic lateral release, subchondral drilling and repair of the MPFL. (B) Postoperative (Post-op) MRI following arthroscopic lateral patellar release (red arrow) and subchondral drilling showing interruption of the low signal subchondral calcified cartilage (white arrows). (C) At one year following surgery, MRI revealed a healed lateral retinaculum (red arrow), re-establishment of the subchondral calcified cartilage (white arrows) and evidence of articular cartilage regeneration at the lateral patella facet.

Fig. 46. A 40-year-old woman with patellar dislocation (STIR images at 0.35T). (A) Preoperative MRI with evidence of a delaminating articular cartilage injury and medial patellar femoral ligament (MPFL) injury. The injury was treated with arthroscopic lateral release, subchondral drilling and repair of the MPFL. (B) Postoperative (Post-op) MRI following arthroscopic lateral patellar release (red arrow) and subchondral drilling showing interruption of the low signal subchondral calcified cartilage (white arrows). (C) At one year following surgery, MRI revealed a healed lateral retinaculum (red arrow), re-establishment of the subchondral calcified cartilage (white arrows) and evidence of articular cartilage

regeneration at the lateral patella facet.

Fig. 47. Sagittal PD MRI (0.35T) of patient in Figure 46. (A) Postoperative (Post-op) MRI: chondral lesions at the lateral patella and lateral femoral chondyle (arrows). Note the disruption to the continous low signal calcified cartilage layer at both sites. (B) MRI at one year after surgery showing the re-establishment of the subchondral calcified cartilage layer (long arrows) together with articular cartilage regeneration (short arrows).

The re-establishment of the calcified cartilage layer and healing of the subchondral bone are important MRI features of articular cartilage regeneration in our series. This is accompanied by filling of the chondral defect. Following arthroscopic subchondral drilling, MRI images revealed extensive marrow edema and interruption of the calcified cartilage layer together with the underlying subchondral bone. This is shown as disruption of the low signal subchondral dark line. As the injected PBPC seed the blood clot scaffold in the presence of HA, chondrogenesis is initiated with the formation of chondrocytes which then occupy the drill holes. This process is gradually replaced by bone resulting in subchondral bone repair. Gradual resolution of marrow edema is observed. The subchondral dark line progressively re-appears on MRI scans indicating the re-establishment of the calcified cartilage layer and healing of the subchondral bone (Figures 45 to 47). This evidence is provided from the histology shown on Figures 20 and 21. Depending on whether the lesion is a contained or uncontained lesion, chondrogenesis follows the gradual re-appearance of this subchondral dark line on serial MRI scans (Figures 24 and 26).

#### **7.4.11 Phase I study clinical outcomes**

Since starting clinical trials in 2007, 223 cartilage regeneration cases have been performed on 205 patients. Cases were varied including 38 cases involving isolated cartilage lesions, 92 cases involving multiple cartilage lesions, 54 cases involving patellofemoral cartilage lesions, 7 cases involving concomitant lower limb realignment procedures, and 32 cases involving ligament reconstruction. When evaluating two-year clinical outcomes, 24 months has passed for 155 of these 223 cases. Within this group, 52 cases have preoperative, 12-month, and 24-month IKDC values available for clinical outcome evaluation. This group had a preoperative IKDC average of 50.5, a 12-month IKDC

Articular Cartilage Regeneration with Stem Cells 173

above baseline and remained stable in the refined method at 25 points above baseline (Zeifang et al, 2010). This compares to our rise above a baseline IKDC of 50.5 by 20 points at 12 months, 20 points at 24 months, and 21 points at 30 months (Fig 48). Of note, our patient group has a mean age of 47.1 years and included patients with multiple chondral lesions,

Our clinical results indicate that the regenerated tissue is resilient and coincide with histological results suggesting that this technique produces hyaline cartilage. However, evaluating phase I data has weaknesses. As this portion of the clinical trial has included evolution in technique and cell processing, there is some inherent variation. Additionally, IKDC collection was inconsistent in the initial phases. Also, when attempting to evaluate this early data, there is no control group available for comparison. Currently a randomized

Specific complications are mild bone pain associated with Neupogen injections, and discomfort of PBSC harvesting and localized pain following intraarticular injections with PBPC and HA. One male patient in his mid-forties had a previous infection from Anterior Cruciate Ligament (ACL) surgery, multiple microfractures and subsequent ACL revision surgery with postoperative PBPC and HA. He had recurrence of intercondylar osteophytes and reactive arthritis and eventually chose a total knee replacement. Three other patients had secondary procedure for persistent osteophytes and treatment for further areas of

It is evident that with adjuvant PBPC and HA therapy, chondrogenesis is possible with hyaline cartilage, but a small proportion of patients may return for further treatment

A randomized controlled trial comparing a group with PBPC and HA injections with a group with HA injection alone following arthroscopic subchondral drilling is currently under way, being supervised by the first author. The early results seem to support the benefit of adjuvant postoperative intraarticular injections of PBPC in combination with

Our theory is that providing a high percentage of immature multipotent progenitor cells into the right environment allows these cells to populate areas of subchondral drilling and regenerate hyaline cartilage. Our histologic findings of chondrocytes below the calcified cartilage layer at a subchondral drill hole (Figs 20 and 21) and the porcine model (Lee et al, 2007) illustrating mesenchymal stem cells at the base of newly formed cartilage support the idea that injected progenitor cells are attracted to the site of marrow injury, proliferate into chondrocytes, and regenerate hyaline cartilage from the subchondral base. We theorize that the addition of matrix substance, in the form of HA and passive stimulating kinetic movement of the involved joint (continuous passive motion), provides chemical and cellular signals for regeneration. Partial to full weight-bearing in the early phase of rehabilitation provides the essential environment to assist in the remodelling of the collagen fibrils to align

because of newly diagnosed areas of chondral degeneration in the treated knee.

while Zeifang et al. (2010) evaluated isolated defects of the femoral condyle.

controlled trial is underway under the direction of the first author.

**7.4.12 Complications**

chondral degeneration.

**7.6 Summary of theory**

HA.

**7.5 Current control randomized trial**

along the axis of weight transmission.

average of 70, and a 24-month IKDC average of 70. 30 patients had 30-month data available and illustrated a 30-month IKDC average of 71 (Fig 48).

Fig. 48. International Knee Documentation Committee (IKDC) outcomes.

Our IKDC results illustrated a significant increase similar to studies documenting overall outcomes in the literature with marrow stimulation and chondrocyte implantation. Historically, microfracture has shown a peak functional outcome at 24 months (Blevins et al, 1998; Peterson et al, 2000; Steadman et al, 2003; Gobbi et al, 2005, Mithoefer et al, 2005; Knutsen et al, 2007). Two studies have illustrated a decline after the first 18 to 24 months with microfracture, including Mithoefer et al (2005) documenting 69% of their patients reporting lower IKDC scores after 24 months. Conversely, two outcome studies have found sustained improvement at the 24-month time interval (Steadman et al, 2003; Kneutsen et al, 2004). In comparison of these four studies, the average age of the study groups documenting decline was 39.5 years and 41 years. In contrast, the average age of the studies documenting sustained improvement was 30.4 years and 31.1 years (Steadman et al, 2003; Mithoefer et al, 2005; Kruez et al, 2006; Knutsen et al, 2004). In a group with an average age of 47.1 years, our IKDC scores showed sustained improvement at 24 months with 30 cases documenting continued improvement at 30 months.

Comparing microfracture and chondrocyte implantation, decisive superiority has not been established. Two randomized studies have sought to directly compare outcomes with differing results. Saris et al. (2009) found significantly better outcomes with ACI at 36 months. They found a continued increase in Knee Injury and Osteoarthritis Outcome Scores (KOOS) from 6 to 36 months with ACI and a plateau in scores at 18 months with microfracture (Saris et al, 2009). However, Knutsen et al. found no significant clinical or radiographic difference between microfracture and ACI at 60 months utilizing four clinical outcome scoring systems and 2 radiologic outcome systems (Knutsen et al,2007). Zeifang et al. (2010) in comparison of conventional methods of ACI with refined methods of ACI for treatment of femoral chondral lesions found an increase from a baseline IKDC (51.1) by 21 points and 25 points respectively. This series had an average age of 29.3 years (Zeifang et al, 2010). At 24 months, this rise declined slightly in the conventional method to 19 points above baseline and remained stable in the refined method at 25 points above baseline (Zeifang et al, 2010). This compares to our rise above a baseline IKDC of 50.5 by 20 points at 12 months, 20 points at 24 months, and 21 points at 30 months (Fig 48). Of note, our patient group has a mean age of 47.1 years and included patients with multiple chondral lesions, while Zeifang et al. (2010) evaluated isolated defects of the femoral condyle.

Our clinical results indicate that the regenerated tissue is resilient and coincide with histological results suggesting that this technique produces hyaline cartilage. However, evaluating phase I data has weaknesses. As this portion of the clinical trial has included evolution in technique and cell processing, there is some inherent variation. Additionally, IKDC collection was inconsistent in the initial phases. Also, when attempting to evaluate this early data, there is no control group available for comparison. Currently a randomized controlled trial is underway under the direction of the first author.

#### **7.4.12 Complications**

172 Modern Arthroscopy

average of 70, and a 24-month IKDC average of 70. 30 patients had 30-month data

**Pre Op 12 24 30 Months**

Our IKDC results illustrated a significant increase similar to studies documenting overall outcomes in the literature with marrow stimulation and chondrocyte implantation. Historically, microfracture has shown a peak functional outcome at 24 months (Blevins et al, 1998; Peterson et al, 2000; Steadman et al, 2003; Gobbi et al, 2005, Mithoefer et al, 2005; Knutsen et al, 2007). Two studies have illustrated a decline after the first 18 to 24 months with microfracture, including Mithoefer et al (2005) documenting 69% of their patients reporting lower IKDC scores after 24 months. Conversely, two outcome studies have found sustained improvement at the 24-month time interval (Steadman et al, 2003; Kneutsen et al, 2004). In comparison of these four studies, the average age of the study groups documenting decline was 39.5 years and 41 years. In contrast, the average age of the studies documenting sustained improvement was 30.4 years and 31.1 years (Steadman et al, 2003; Mithoefer et al, 2005; Kruez et al, 2006; Knutsen et al, 2004). In a group with an average age of 47.1 years, our IKDC scores showed sustained improvement at 24 months with 30 cases documenting

Comparing microfracture and chondrocyte implantation, decisive superiority has not been established. Two randomized studies have sought to directly compare outcomes with differing results. Saris et al. (2009) found significantly better outcomes with ACI at 36 months. They found a continued increase in Knee Injury and Osteoarthritis Outcome Scores (KOOS) from 6 to 36 months with ACI and a plateau in scores at 18 months with microfracture (Saris et al, 2009). However, Knutsen et al. found no significant clinical or radiographic difference between microfracture and ACI at 60 months utilizing four clinical outcome scoring systems and 2 radiologic outcome systems (Knutsen et al,2007). Zeifang et al. (2010) in comparison of conventional methods of ACI with refined methods of ACI for treatment of femoral chondral lesions found an increase from a baseline IKDC (51.1) by 21 points and 25 points respectively. This series had an average age of 29.3 years (Zeifang et al, 2010). At 24 months, this rise declined slightly in the conventional method to 19 points

available and illustrated a 30-month IKDC average of 71 (Fig 48).

Fig. 48. International Knee Documentation Committee (IKDC) outcomes.

continued improvement at 30 months.

**Increase in IKDC**

Specific complications are mild bone pain associated with Neupogen injections, and discomfort of PBSC harvesting and localized pain following intraarticular injections with PBPC and HA. One male patient in his mid-forties had a previous infection from Anterior Cruciate Ligament (ACL) surgery, multiple microfractures and subsequent ACL revision surgery with postoperative PBPC and HA. He had recurrence of intercondylar osteophytes and reactive arthritis and eventually chose a total knee replacement. Three other patients had secondary procedure for persistent osteophytes and treatment for further areas of chondral degeneration.

It is evident that with adjuvant PBPC and HA therapy, chondrogenesis is possible with hyaline cartilage, but a small proportion of patients may return for further treatment because of newly diagnosed areas of chondral degeneration in the treated knee.

#### **7.5 Current control randomized trial**

A randomized controlled trial comparing a group with PBPC and HA injections with a group with HA injection alone following arthroscopic subchondral drilling is currently under way, being supervised by the first author. The early results seem to support the benefit of adjuvant postoperative intraarticular injections of PBPC in combination with HA.

#### **7.6 Summary of theory**

Our theory is that providing a high percentage of immature multipotent progenitor cells into the right environment allows these cells to populate areas of subchondral drilling and regenerate hyaline cartilage. Our histologic findings of chondrocytes below the calcified cartilage layer at a subchondral drill hole (Figs 20 and 21) and the porcine model (Lee et al, 2007) illustrating mesenchymal stem cells at the base of newly formed cartilage support the idea that injected progenitor cells are attracted to the site of marrow injury, proliferate into chondrocytes, and regenerate hyaline cartilage from the subchondral base. We theorize that the addition of matrix substance, in the form of HA and passive stimulating kinetic movement of the involved joint (continuous passive motion), provides chemical and cellular signals for regeneration. Partial to full weight-bearing in the early phase of rehabilitation provides the essential environment to assist in the remodelling of the collagen fibrils to align along the axis of weight transmission.

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*1.1 Definition*

According to the World Health Organization definition, cardiac rehabilitation is "the sum of activities required to influence, favorably, the underlying cause of the disease, as well as to provide the best possible physical, mental, and social conditions, so that patients may, by their own efforts, preserve or resume optimal functioning in their community and through improved health behavior, slow or reverse the progression of a disease" [1,2]. The objectives of exercise-based cardiac rehabilitation are to increase functional capacity level, reduce anginal symptoms and disability, improve quality of life, modify coronary risk factors, and reduce morbidity and mortality rates [3].

Key elements of the comprehensive cardiac rehabilitation program include [3,4]:

Exercise prescription remains a core component of cardiac rehabilitation.

A significant reduction in cardiovascular mortality and hospitalization has recently been validated by Cochrane analysis. The beneficial effects of cardiac rehabilitation reported in a group of patients after myocardial infarction include reductions in [5–7]:

More recent Cochrane review demonstrated a significant reduction in cardiovascular mortality in exercising group compared with controls (10.4% and 7.6% respectively) [8].

The specific mechanisms responsible for these beneficial effects remain disputable, especially considering the insignificant reduction in the recurrence of myocardial infarction after cardiac rehabilitation. It has been postulated that mortality reduction is a result of a reduction in ventricular fibrillation (decreased sympathetic tone and enhanced parasympathetic tone) or due to the mechanism of ischemic preconditioning [9]. As cardiorespiratory fitness improvement is associated with reductions in mortality after a structured, comprehensive cardiac rehabilitation program, the exact mean change in fitness that occurs has been extensively studied. Sandercock's review of 31 studies demonstrated an increase in fitness of 1.55 metabolic equivalents after phase II. Converting this value to peak oxygen uptake, a gain in cardiorespiratory fitness of 5.4 mL/kg/min can be expected. The reported extent of gain was strictly related to the number of sessions completed, with >36 exercise sessions resulting in a greater gain in fitness [10]. As expected, programs with a significantly lower number of sessions resulted in a smaller increase in cardiorespiratory fitness [11].

The effectiveness of cardiac rehabilitation in reducing either cardiovascular mortality or the rate of myocardial re-infarction largely depends on the exercise volume. Data from meta-analyses have demonstrated specific requirements for the best cardiac rehabilitation outcomes as presented in Table 1 [12].

**Table 1.** Optimal conditions for efficacious cardiac rehabilitation.

Source: Authors compilation based on data from [12].

### *1.2. Development*

In the 18th century, Heberden reported that exertion reduced the frequency of anginal pain. He described a patient's health improvement after sawing wood for 30 min every day [13]. The practice of imposing limited mobility on patients with acute coronary events, however, continued over the next 150 years, leading to prolonged hospital stays and declines in functional capacity [14]. In the 1930s, patients remained bedridden for 6 weeks following myocardial infarction due to the pathologic finding that the infarction evolution process from initial ischemic necrosis to the formation of a stable scar lasts over 6 weeks. Chair therapy was introduced in the 1940s, and, from the early 1950s, a strategy of very short daily walks lasting five minutes was advocated, but only after a coronary event and four subsequent weeks of rest [15]. In 1968, a study by Saltin demonstrated the importance of exercise and the detrimental effect of prolonged bed rest [16]. Further studies by Braunwald, Hutter, Askanas, Rudnicki, Boyle, Sonnenblick, Hellerstein, Naughton, and many others established a strong case for the benefits of exercise and supported the use of early mobilization in hospital settings [17]. As a result, a phasic, multi-profile model of cardiac rehabilitation has gradually been implemented, including a return to work and social activities [18]. Early ambulation has become standard and evolved into phase 1 or inpatient cardiac rehabilitation. Activities performed in the coronary intensive care unit are typically limited to 2 METs and include self-care (such as bed bathing, sitting in a chair) and passive and active ranges of motion. The further rapid development of the use of interventional techniques in cardiology, however, has significantly shortened the duration of hospitalization following an acute coronary event to 3–5 days, thus allowing for more intense mobilization processes to be used.

Contemporary cardiac rehabilitation has gradually evolved into a comprehensive secondary prevention, multi-factorial program consisting of exercise training; the management of cardiovascular risk factors; and nutritional, psychological, behavioral, and social support to improve patient outcomes. The target population for cardiac rehabilitation has expanded significantly over the years, but post-myocardial infarction survivors remain a key population. In the guidelines of the European Society of Cardiology and the American Heart Association, cardiac rehabilitation is mandatory—i.e., it has class I recommendations for therapeutic intervention in many cardiac conditions (e.g., acute coronary syndromes, percutaneous coronary interventions and/or myocardial revascularization surgery, stable coronary artery disease, heart failure, cardiac transplant, left ventricular assistance devices or other implanted devices (cardiac pacemakers, cardioverter-defibrillators, cardiac resynchronization devices), cardiac surgery, and high cardiovascular risk-factor profiles) [19]. The different classes of recommendations, with the corresponding level of evidence-based indications needed for the most frequently referred groups of patients, are listed in Table 2.

**Table 2.** Evidence-based indications for cardiac rehabilitation.

Source: Table by authors.

Cardiac rehabilitation is constantly evolving, with new ideas and strategies being implemented all the time. Examples of new strategies are, hybrid telerehabilitation and high-intensity interval training. Recent COVID 19 pandemic highlighted the role of cardiac telerehabilitation as an efficacious, safe, and essential part of cardiac rehabilitation. There is still, however, a need to enhance cardiac rehabilitation enrollment due to the lower cardiac rehabilitation referral and participation rates of women, the elderly, and minorities [20–22]. Furthermore, cardiac rehabilitation is offered in just 55% of countries, as exhibited in Figure 1 [23].

Figure 1. Global cardiac rehabilitation availability. Source: Reprinted from [23]. **Figure 1.** Global cardiac rehabilitation availability. Source: Reprinted from [23].

Thus, the distribution of cardiac rehabilitation services to the highest possible number of eligible patients should be prioritized, as should overcoming many existing barriers—e.g., the insufficient education of patients, poor adherence, lack of availability of structured programs, etc.


**1. Introduction**

In its simplest sense, the environment means the surrounding external conditions influencing the growth of people, animals or plants, living or working conditions, etc. Environmental Sciences (EVS) is an integrated multidisciplinary approach that studies the environment and solutions of environmental problems. In the present scenario, the environment has become a global agenda item, which has increased the scope and importance of EVS. In the development of different stages of civilization, humans were accompanied both by the environment and statistics. Since the early days, they were found to be knowingly accustomed to the environment and unknowingly played with statistics. Thus, both statistics and the environment have shared a long history of mutual reciprocation. In modern times, these two subjects are independently able to attract the academic attention of scholars throughout the world (see [1]).

The United Nations Statistics Division (UNSD) has an exclusive branch for environmental statistics, established in 1995. Its major area of work is data collection, methodology, capacity development, and coordination of environmental statistics and indicators. They have a dedicated newsletter called "ENVSTATS", which publishes the activities of UNSD in the area of environmental statistics. The Framework for the Development of Environmental Statistics (FDES 2013) is an updated version of the original FDES, which was published by UNSD in 1984. In India, the Ministry of Statistics and Programme Implementation has a specific publication report in the branch of environmental statistics called "EnviStats" which updates recent developments in the field of environmental statistics.

The extensive use of statistics in EVS led to the development of a new branch called Environmental Statistics. We all know that statistics are an inevitable context in any scientific arena. Even so, the motivation for conducting this specific review is that environmental statistics have an integrated multidisciplinary face, which will shed light on the pure biological field of modern science with its analytical nature. That undiscovered interconnection with statistics and environmental science will be revealed through this review, which will be an easy access point for future investigators. This review has been conducted in two

**Citation:** Tomy, L.; Chesneau, C.; Madhav, A.K. Statistical Techniques for Environmental Sciences: A Review. *Math. Comput. Appl.* **2021**, *26*, 74. https://doi.org/10.3390/ mca26040074

Received: 8 October 2021 Accepted: 1 November 2021 Published: 4 November 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

5

parts, i.e., the pure statistical techniques and those specific techniques that have been exclusively invented for environmental science. A brief state of the art is presented below. The authors of [2] discussed different statistical techniques which are helpful to environmental engineers. It addresses different environmental problems with a solution-oriented approach that encourages students to view statistics as a problem-solving tool.

The use of statistical techniques to understand various environmental phenomena was explained in [3]. He examined different statistical tools, such as probabilistic and stochastic models, data collection, data analysis, inferential statistics, etc. In addition, he discussed principles and methods applicable to a wide range of environmental issues (including pollution, conservation, management, control, standards, sampling, monitoring, etc.) across all fields of interest and concern (including air and water quality, forestry, radiation, climate, food, noise, soil condition, fisheries and environmental standards). Accordingly, he considered sophisticated statistical techniques, such as extreme processes, stimulus response methodology, linear and generalized linear models, sampling principles and methods, time series, spatial models, multivariate techniques, design of experiments, etc.

This article is an attempt to describe some basic statistical concepts used in EVS, thereby establishing a link between the two subjects. It studies some basic statistical concepts relevant to environmental study. Illustrations are discussed on the basis of [4,5].

In this article, Section 2 presents the basic concepts in statistics, Section 3 describes the application of statistical tools in EVS, Section 4 is about the various illustrations regarding the topic, and Section 5 is the conclusion.

## **2. Basic Concepts**

With the advent of the theory of probability and games of chance in the mid-seventeenth century, the concept of modern statistics was born. The name "statistics" appears to have come from the German word "Statistik," the Italian word "statista," or the Latin word "status," all of which mean "political state" or "state craft", respectively. The term statistics can be used in two different senses.

In the plural sense, it means "a collection of numerical facts". According to Horace Secrist, "Statistics may be defined as the aggregate of facts, affected to a marked extent by a multiplicity of causes, numerically expressed, enumerated or estimated according to a reasonable standard of accuracy, collected in a systematic manner, for a predetermined purpose and placed in relation to each other". This definition explains the characteristics of statistical data.

In its singular sense, it means "statistical methods for dealing with numerical data". According to Croxton and Cowden, "Statistics is the science of collection, presentation, analysis, and interpretation of numerical data". This definition points out different stages of statistical investigation. Hence, statistics is concerned with exploring, summarizing, and making inferences about the state of complex systems, for example, the state of a nation (social statistics), the state of people's health (medical and health statistics), the state of the environment (environmental statistics), as extensively described in [6].

In the midst of its wide range of applications and advantages, one important allegation about statistics is that the concerned parties may make misleading statements in their favor. However, the fact is that, as in the case of any science, only an expert can make use of statistical tools effectively. One should make sure that the statistical study is conducted by the right person. There are lots of good ways, many more bad and wrong ways too. So, be sure about the correctness of the tool used. The notorious allegation by Mark Twain citing the British Prime Minister Benjamin Disraeli that "there are three types of lies: lies, damned lies, and statistics" (but the phrase is nowhere in Disraeli's works, and the earliest known appearances were years after his death, so it is assumed to be by some anonymous writer in mid-1891) is just a lie, provided the precaution is served. In such a context, it is interesting that the author of [7] beautifully coined the title "Truth, Damn Truth and Statistics" for his article.

## **3. Application of Statistical Tools in EVS**

In statistics, data analysis is divided into two sections: descriptive statistics and inferential statistics. The authors of [5] discussed the two in depth, and Sections 3.1 and 3.2 below present them in summary form.

#### *3.1. Descriptive Statistics*

Descriptive statistics are the initial stage of data analysis where exploration, visualization and summarization of data are done. We will look at the definitions of population and random sample in this section. Different types of data, viz. quantitative or qualitative, discrete or continuous, are helpful for studying the features of the data distribution, patterns, and associations. The frequency tables, bar charts, pie diagrams, histograms, etc., represent the data distribution, position, spread and shape efficiently. This descriptive statistical approach is useful for interpreting the information contained in the data and, hence, for drawing conclusions.

Further, different measures of central tendency viz. mean, median, etc., were calculated for analyzing environmental data. It is also useful to study dispersion measures, such as range, standard deviation, etc., to measure variability in small samples. One of the important measures of relative dispersion is the coefficient of variation, and it is useful for comparing the variability of data with different units. Skewness and kurtosis characterize the shape of the sample distribution. The concepts of association and correlation demonstrate the relationships between variables and are useful tools for a clear understanding of linear and non-linear relationships. Important measures of these fundamental characteristics are briefly discussed here in the following.

#### 3.1.1. Central Tendency

The tendency of the observations to cluster around some central value is called central tendency. Any measure of central tendency is termed "average". The most commonly used averages are the following:

$$\text{Mean } \mathfrak{x} = \sum\_{i=1}^{n} \frac{x\_i}{n}.$$

where *xi* denotes the *i*th observation and *n* is the number of observations.

Median is the middle-most observation when observations are arranged in ascending or descending order

and

Mode is the most frequently occurring observation.

#### 3.1.2. Dispersion

The scattering of observations about the central value is called dispersion. Important measures of dispersion are range, quartile deviation, mean deviation, standard deviation and coefficient of variation. These four measures depend on the unit of measurement of the observations, hence, they are absolute measures. They can be defined as:

Range is the difference between largest and smallest observations.

Measure based on quartiles:

• Quartile deviation

$$QD = \frac{Q\_3 - Q\_2}{2}$$

where *Q*3 and *Q*2 are the third and first quartile in the frequency distribution, respectively;

• Mean deviation

$$MD = \sum\_{i=1}^{n} \frac{|\mathbf{x}\_i - \vec{x}|}{n}$$

where *x*¯ is mean of *xi* (observed values);

• Standard deviation

$$SD = \sqrt{\sum\_{i=1}^{n} \frac{(\mathbf{x}\_i - \overline{\mathfrak{x}})^2}{n}};$$

• Coefficient of variation

$$CV = \frac{SD}{\text{Mean}} \times 100.$$

Thus, CV is the relative measure (measure independent of unit) corresponding to SD.

3.1.3. Skewness

The lack of symmetry is termed as skewness or asymmetry. In a frequency curve, if both the sides of the mode are distributed in the same manner, the distribution is symmetric, otherwise it is skewed. When more area is on the right side of the mode, the distribution is positively skewed. If more area is on the left side of the mode, the distribution is negatively skewed. Figure 1 depicts the three situations. There are mainly two measures:

1. Pearson's measure

$$\mathcal{S} = \frac{\text{Mean} - \text{Mode}}{SD}$$

If *S* = 0, the distribution is symmetric, if *S* > 0, positively skewed and if *S* < 0, negatively skewed.

2. Moment measure

$$\beta\_1 = \frac{\mu\_3}{\mu\_2^{3/2}}$$

*μ*2 = *SD*2

where

and

$$\mu\_3 = \sum\_{i=1}^n \frac{(x\_i - \bar{x})^3}{n}.$$

If *β*1 = 0, it is symmetric, if *β*1 > 0, positively skewed and if *β*1 < 0, negatively skewed.

**Figure 1.** (**a**) Negative skewness, (**b**) symmetric, and (**c**) positive skewness.


**2. Nutrition deficiency and related challenges**

With the increasing world population, enhancing the production of food and nutritional quality of staple crops is the strategy to address the emerging food crises [7]. A food crisis causes multidimensional effects on human nutrition, and it causes malnutrition. It also has effects on the supply of food quantity and quality of food. In the last two decades, these problems have been tried to be solved to reduce the proportion of the world's malnourished population [8]. Protein deficiency malnutrition has emerged as a major nutritional problem, particularly in the developing countries [9]. In the developing countries, cereals play an important source of dietary protein for humans, which comprise 70% of the protein intake [10]. Maize is the world's third primary cereal crop, which is an important protein source used as food and feed for humans and animals and also used in corn starch industry, corn oil production, etc. [11]. QPM has more quantity of carbohydrates, fats, proteins, vitamins and minerals. It is also called as a 'poor man's cereal crop'. In developing countries like Africa and Latin America, as the animal protein is very limited and expensive, which results in being unavailable to a vast sector of the population, maize grains provide about 15–56% of total daily calories in people's diets [12]. Nearly 9.09 million hectares were allocated to cultivate maize, which produces nearly 24.26 million tons in India and can be cultivated throughout the year [13]. Maize proteins consists just 1.81 and 0.35% of lysine and tryptophan content, respectively, which is very low compared with the Food and Agriculture Organization (FAO) recommendation. From the human nutrition perspective, lysine and tryptophan are the most considerable limiting amino acid in the maize endosperm protein. Thus humans and other monogastric animals should include other alternative sources of lysine and tryptophan in their healthy diets [14]. Babies fed on normal maize without any protein supplements suffer from malnutrition and develop Kwashiorkor disease [15]. In this context, the International Maize and Wheat Improvement Center (CIMMYT) and the International Institute of Tropical Agriculture (IITA) are developing varieties to improve the protein quality of maize by incorporating the opaque-2, along with modifier genes, thus increasing the amount of lysine (>4.0%) and tryptophan (>0.8%) contents in the whole grain compared with normal maize [16]. Maize cultivars containing high yield with increasing levels of lysine and tryptophan and having the kernel structure of conventional maize have the potential to reduce the malnutrition [14].

## **3. Storage proteins in QPM**

The mature maize kernel consists of a germ, pericarp and endosperm. An endosperm consists of 90% starch which is a source of concentrated energy and 10% protein which include albumins, globulins, zein and glutelin out of which zein consists 50–70% of total proportion [10]. Zeins are the important storage proteins; these forms as deposit on rough endoplasmic reticulum-delimited protein bodies (PBs) [17]. During the maturation of kernel, these protein bodies become densely packed between starch grains in the vitreous regions of the endosperm [18]. Zeins

**35**

*DOI: http://dx.doi.org/10.5772/intechopen.89038*

**3.1 Zein gene**

22,000 gene promoters [23].

in the endosperm development.

**4. Nutrition analysis of QPMs**

*Quality Protein Maize: An Alternative Food to Mitigate Protein Deficiency in Developing…*

are a group of four structurally distinct alcohol-soluble proteins (α-zein, β-zein, γ-zein and δ-zein) [17] present only in seeds' endosperm and playing a key role in storing and supplying N, C and S to the germinating seedling [12]. Among those zein proteins, α-zeins and δ-zeins are deposited in the central region, and γ-zeins and β-zeins were deposited in the outer region of protein bodies [19]. The zein fractions are rich in cysteine and methionine amino acids, and it also consists of glutamine, leucine and proline and is completely devoid of two important essential amino acids lysine and tryptophan, whereas other proteins consist of these amino acids in large quantities [20]. The zein synthesis serves as a model system to study coordinated genetic regulation of several genes expressed at very high levels at a specific developmental stage. Suppression of zein fraction without drastically altering the contribution of other fractions could be, thus, seen as a feasible approach to

bring about improvements in the amino acid balance in maize grain [12].

Zein is a class of prolamin proteins that are mainly present in maize. All the zein polypeptides are products of different structural genes [21]. Most of the prolamin genes have a promoter element called the endosperm or prolamin box. The promoter element is present about the 300 base pairs upstream of the translation start codon and has a conserved 15-bp element that contains the 7-bp endosperm motif (TGTAAAG) [22]. This endosperm motif acts as a tissue-specific enhancer in Mr.

Genetic analysis of o2 modifiers revealed several quantitative trait loci (QTLs) dispersed on the chromosomes. These identified QTLs were correlated with the 27-kDa γ-zein gene expression and protein quantity in QPM [24]. The 27-kDa γ-zein gene expression is not under the control of the o2 protein [25]. The o2 modifier genes involved in the 27-kDa γ-zein gene expressions are observed in two different QTLs. The first of these is associated with increased expression [26]. Single copy of γ-zein genes encodes the 50, 27 and 16-kDa proteins, which were observed in the B73 genome [27]. Based on the allotetraploidization and protein-sequence similarity, both 27 and 16 kDa γ-zein genes originated from a common progenitor [28]. It is about 20–25% of total zeins; the low abundance 50-kDa γ-zein gene has low similarity with other two γ-zein genes [27]. The γRNAi and βRNAi were involved in maize kernel opacity to increase the intensification. It reveals that opacity was not involved in reducing the thickness of the opaque-2-mutated endosperm; it is due to partial arrangement of starch granules in the endosperm [29]. Although discrete protein bodies were observed in endosperm cells, honeycomb-like masses of protein bodies were observed. It indicates that different zeins have played an important role

Generally, quality of protein nutrition was estimated by composition of amino

acids, digestibility and amino acid requirement to consume the protein. The QPMs are reported to have increased levels of lysine and tryptophan in the endosperm protein, which enhances the biological value of protein similar to the milk protein. It has brought about great hope in the effort to improve human nutrition [30]. Firstly there is a significant difference in the QPM kernel when compared to normal maize kernel. Kernel hardness was determined by calculating floatation index where it is 57% for QPM, whereas for normal maize, it is 19.7%. The whole kernel protein was 13.15% in QPMs with contribution of 8.6 and 13.88% from

*DOI: http://dx.doi.org/10.5772/intechopen.89038 Quality Protein Maize: An Alternative Food to Mitigate Protein Deficiency in Developing…*

are a group of four structurally distinct alcohol-soluble proteins (α-zein, β-zein, γ-zein and δ-zein) [17] present only in seeds' endosperm and playing a key role in storing and supplying N, C and S to the germinating seedling [12]. Among those zein proteins, α-zeins and δ-zeins are deposited in the central region, and γ-zeins and β-zeins were deposited in the outer region of protein bodies [19]. The zein fractions are rich in cysteine and methionine amino acids, and it also consists of glutamine, leucine and proline and is completely devoid of two important essential amino acids lysine and tryptophan, whereas other proteins consist of these amino acids in large quantities [20]. The zein synthesis serves as a model system to study coordinated genetic regulation of several genes expressed at very high levels at a specific developmental stage. Suppression of zein fraction without drastically altering the contribution of other fractions could be, thus, seen as a feasible approach to bring about improvements in the amino acid balance in maize grain [12].

#### **3.1 Zein gene**

*Maize - Production and Use*

maize' [3].

modifier genes, which present hard endosperm in the o2 background, were developed at the International Maize and Wheat Improvement Center (CIMMYT), Mexico [5], and University of Natal, South Africa [6]. This leads to the development of nutritionally enriched hard endosperm maize, widely known as 'quality protein

With the increasing world population, enhancing the production of food and nutritional quality of staple crops is the strategy to address the emerging food crises [7]. A food crisis causes multidimensional effects on human nutrition, and it causes malnutrition. It also has effects on the supply of food quantity and quality of food. In the last two decades, these problems have been tried to be solved to reduce the proportion of the world's malnourished population [8]. Protein deficiency malnutrition has emerged as a major nutritional problem, particularly in the developing countries [9]. In the developing countries, cereals play an important source of dietary protein for humans, which comprise 70% of the protein intake [10]. Maize is the world's third primary cereal crop, which is an important protein source used as food and feed for humans and animals and also used in corn starch industry, corn oil production, etc. [11]. QPM has more quantity of carbohydrates, fats, proteins, vitamins and minerals. It is also called as a 'poor man's cereal crop'. In developing countries like Africa and Latin America, as the animal protein is very limited and expensive, which results in being unavailable to a vast sector of the population, maize grains provide about 15–56% of total daily calories in people's diets [12]. Nearly 9.09 million hectares were allocated to cultivate maize, which produces nearly 24.26 million tons in India and can be cultivated throughout the year [13]. Maize proteins consists just 1.81 and 0.35% of lysine and tryptophan content, respectively, which is very low compared with the Food and Agriculture Organization (FAO) recommendation. From the human nutrition perspective, lysine and tryptophan are the most considerable limiting amino acid in the maize endosperm protein. Thus humans and other monogastric animals should include other alternative sources of lysine and tryptophan in their healthy diets [14]. Babies fed on normal maize without any protein supplements suffer from malnutrition and develop Kwashiorkor disease [15]. In this context, the International Maize and Wheat Improvement Center (CIMMYT) and the International Institute of Tropical Agriculture (IITA) are developing varieties to improve the protein quality of maize by incorporating the opaque-2, along with modifier genes, thus increasing the amount of lysine (>4.0%) and tryptophan (>0.8%) contents in the whole grain compared with normal maize [16]. Maize cultivars containing high yield with increasing levels of lysine and tryptophan and having the kernel structure of

conventional maize have the potential to reduce the malnutrition [14].

The mature maize kernel consists of a germ, pericarp and endosperm. An endosperm consists of 90% starch which is a source of concentrated energy and 10% protein which include albumins, globulins, zein and glutelin out of which zein consists 50–70% of total proportion [10]. Zeins are the important storage proteins; these forms as deposit on rough endoplasmic reticulum-delimited protein bodies (PBs) [17]. During the maturation of kernel, these protein bodies become densely packed between starch grains in the vitreous regions of the endosperm [18]. Zeins

**2. Nutrition deficiency and related challenges**

**34**

**3. Storage proteins in QPM**

Zein is a class of prolamin proteins that are mainly present in maize. All the zein polypeptides are products of different structural genes [21]. Most of the prolamin genes have a promoter element called the endosperm or prolamin box. The promoter element is present about the 300 base pairs upstream of the translation start codon and has a conserved 15-bp element that contains the 7-bp endosperm motif (TGTAAAG) [22]. This endosperm motif acts as a tissue-specific enhancer in Mr. 22,000 gene promoters [23].

Genetic analysis of o2 modifiers revealed several quantitative trait loci (QTLs) dispersed on the chromosomes. These identified QTLs were correlated with the 27-kDa γ-zein gene expression and protein quantity in QPM [24]. The 27-kDa γ-zein gene expression is not under the control of the o2 protein [25]. The o2 modifier genes involved in the 27-kDa γ-zein gene expressions are observed in two different QTLs. The first of these is associated with increased expression [26]. Single copy of γ-zein genes encodes the 50, 27 and 16-kDa proteins, which were observed in the B73 genome [27]. Based on the allotetraploidization and protein-sequence similarity, both 27 and 16 kDa γ-zein genes originated from a common progenitor [28]. It is about 20–25% of total zeins; the low abundance 50-kDa γ-zein gene has low similarity with other two γ-zein genes [27]. The γRNAi and βRNAi were involved in maize kernel opacity to increase the intensification. It reveals that opacity was not involved in reducing the thickness of the opaque-2-mutated endosperm; it is due to partial arrangement of starch granules in the endosperm [29]. Although discrete protein bodies were observed in endosperm cells, honeycomb-like masses of protein bodies were observed. It indicates that different zeins have played an important role in the endosperm development.

## **4. Nutrition analysis of QPMs**

Generally, quality of protein nutrition was estimated by composition of amino acids, digestibility and amino acid requirement to consume the protein. The QPMs are reported to have increased levels of lysine and tryptophan in the endosperm protein, which enhances the biological value of protein similar to the milk protein. It has brought about great hope in the effort to improve human nutrition [30]. Firstly there is a significant difference in the QPM kernel when compared to normal maize kernel. Kernel hardness was determined by calculating floatation index where it is 57% for QPM, whereas for normal maize, it is 19.7%. The whole kernel protein was 13.15% in QPMs with contribution of 8.6 and 13.88% from

endosperm and germ, whereas it is 9.25% in normal maize with contribution of 7.9 and 1.28% from endosperm and germ, respectively [18]. An improvement of protein quality has been correlated with the presence of the opaque-2 mutant gene [31]. Crude protein of QPM was higher than the normal maize, and the proportional contribution of the germ is lower in QPMs than with normal varieties. These structural and biochemical changes that happen in the kernel lead to the modifications of the protein profile, both in content and structure, and therefore on the functionality of the protein extracted from QPM [30]. Based on the chemical component analysis, QPM whole kernels showed highest protein content compared with normal maize [32].


**1. Introduction**

380 Herbicides – Properties, Synthesis and Control of Weeds

Richardson, B., Davenhill, N., Coker, G., Ray, J., Vanner, A. & Kimberley, M. (1996a).

Richardson, B., Vanner, A., Ray, J., Davenhill, N. & Coker, G. (1996b). Mechanisms of *Pinus*

SAS Institute Inc. (2004). *SAS/STAT 9.1 user's guide, Vols. 1-7* (1st ed.). SAS Institute Inc. Cary,

Shibu, J., Ranasinghe, S. & Ramsey, C.L. (2010). Longleaf pine (*Pinus palustris* P. Mill.)

Thatcher, R. C., Searcy, J. L., Coster, J. E. & Hertel, G. D. (Eds.). (1980). *The Southern Pine*

Service. U.S. Government Printing Office. Pineville, Louisiana.

size. *New Zealand Journal of Forestry Science* 26(1/2): 265-275.

*Journal of Forestry Science* 26(3): 421-437.

446). John Wiley and Sons. New York, NY.

North Carolina.

Optimising spot weed control: first approximation of the most cost-effective spot

*radiata* growth suppression by some common forest weed species. *New Zealand*

restoration using herbicides: overstory and understory vegetation responses on a coastal plain flatwoods site in Florida, U.S.A. *Restoration Ecology* 18(2): 244-251. Stout, I. J. & Marion, W. R. (1993). Pine flatwoods and xeric pine forest of the southern

(lower) coastal plain. In W. H. Martin, S. G. Boyce & A. C. Echternacht (Eds.), *Biodiversity of the southeastern United States, lowland terrestrial communities* (pp. 373-

*Beetle*. (USDA Technical Bulletin 1631). Expanded Southern Pine Beetle Research and Application Program, Science and Education Administration, USDA Forest

Sugar beet occupies each year about 65.000 hectares in Morocco which allows a production that approaches or exceeds three million tons of roots, with an average yield of 46 tonnes per ha (54% of national needs sugar consumption). Since its introduction in Morocco in 1962-1963, sugar beet yield increased significantly in quantity and quality. In Morocco, the sugar beet is a very important crop because of its products and by-products, mainly:

In Morocco, sugar beet is planted from September through June - July. Yield obtained by farmers, averaging 46T/ha, is significantly below the request potential that would be 90 to 100 T/ha. Many factors contribute to low sugar beet production. Poor stand establishment, inadequate weed control, inadequate insect control and inadequate nitrogen fertilization are the main causes of low tonnage and poor quality sugar beet in Morocco.

The sugar beet is an important strategic crop in the irrigated perimeter of Tadla. During these 5 last years, an annual surface of 12000 ha is emblaved by this crop representing 23% / of the national area. The average yield obtained in the region is approximately 45 to 50 T/ha, which is very low compared to the potential yield.

Sanitary problems particularly weed management is a great constraint to sugar beet production and weeds may cause high yield losses (Rzozi et al., 1990). This paper presents the main results of investigations and experiments conducted in Tadla region to improve the weed management program by identifying mains weed species encountered in sugar beet field, studying the effect of weeds on sugar beet growth and estimating yield losses and determining the critical period of weed control and evaluating herbicide treatments.

Sugar Beet Weeds in Tadla Region (Morocco):

in order to characterize soil texture and total calcium content.

2: averagely abundant species, low recovery, irregular distribution 3: abundant species, covering less than 50%, regular distribution 4: abundant species, recovery of 50 to 75%, regular distribution

5: very abundant species, recovery from 75 to 100%, regular distribution

+: Very rare species (1 to 5 feet), virtually no recovery. 1: scarce species, recovery very low, irregular distribution

(Boulet et al, 1984). The methodology was as follows:

**2.2.2 Prospecting and sampling**

index is as follows:

Species Encountered, Interference and Chemical Control 383

A total of 126 sugar beet fields were explored. Only fields not chemically treated and weedy full kept by farmers were prospected. A stratified sampling according to Gounot (1969) was established taking account of some factors mainly type of soil, rainfall and temperatures. Meanwhile, farmers were questioned about cultural practices and soil samples were taken

The method of the "tower field" has been adopted to identify the weed species present (Maillet, 1981), for which an Abundance-Dominance Index (ADI) (+, 1, 2, 3, 4, 5) according to the scale of Montegut (Not dated) modified by Boulet et al. (1989) has been assigned. This

The agronomic importance of each species is judged based on its relative frequency and covering. The estimation of the average abundance of species during the reading was conducted assuming equivalences between the ADI and its average covering in percentage

ADI Covering Average covering

5 75 – 100 87,5 4 50 – 75 65,5 3 25 - 50 37, 5 2 5 – 25 17, 5 1 1 – 5 5 + < 1 1

These values allow calculating the average covering R of each species at reading time. The combination of this index, of the absolute frequency of species and their ethological type, allowed attribution of a "Partial Nuisibility Index" (PNI) to species (Bouhache et al, 1984). PNI = (Sum of coverage/number of reading) x 100. The perennial species are underlined

Species encountered were identified by using some documents such as Flora Europea (Tutin et al., 1964- 1984), Catalogue des Plantes du Maroc (Jahandiez and Maire, 1931-34) and Mauvaises herbes des regions arides et semi arides du Maroc occidental (Tanji et al., 1988). The ethological type for each species was determined according to classification elaborated by Raunkiaer (1905). The biogeographical origin of weed species was derived from Quezel

and only species with a frequency higher than 20% are taken in consideration.

and Santa (1962-63) and Negre (1961-62) on flora investigations.